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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5943_Библиотеки_им_академика_М_И_Перельмана
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2 Recent Development in invivo Clinical Gene Therapy Platforms
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51 Maeder, M.L., Stefanidakis, M., Wilson, C.J. etal. (2019). Development of a
gene‐editing approach to restore vision loss in Leber congenital amaurosis type
10. Nat. Med. 25 (2): 229–233.
52 Mancuso, P., Chen, C., Kaminski, R. etal. (2020). CRISPR based editing of SIV
proviral DNA in ART treated non‐human primates. Nat. Commun. 11 (1): 6065.
53 McGaw, C., Garrity, A.J., Munoz, G.Z. etal. (2022). Engineered Cas12i2 is a
versatile high‐efficiency platform for therapeutic genome editing. Nat. Commun.
13 (1): 2833.
54 Kim, D.Y., Lee, J.M., Moon, S.B. etal. (2022). Efficient CRISPR editing with a
hypercompact Cas12f1 and engineered guide RNAs delivered by adeno‐
associated virus. Nat. Biotechnol. 40 (1): 94–102.
55 Anzalone, A.V., Koblan, L.W., and Liu, D.R. (2020). Genome editing with
CRISPR‐Cas nucleases, base editors, transposases and prime editors. Nat.
Biotechnol. 38 (7): 824–844.
Section II
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Translational Biomarkers forGene Therapy
61
3
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Biomarker and Bioanalytical Readouts for the
Development of AAV Gene Therapy
Yanmei Lu1 and Wibke Lembke
1
Biomarker and BioAnalytical Sciences, Sangamo Therapeutics, Richmond, CA, USA
2
Bioanalytical Services, Celerion Switzerland AG, Allmendstrasse 32, 8320 Fehraltorf, Switzerland
2
3.1 Introduction
3.1.1 AAV-Mediated invivo Gene Therapy
63
In vivo gene therapy (GTx) has been proven to be a viable therapeutic approach
and holds enormous potential to treat patients with rare diseases with monogenic
mutations as well as complex acquired disorders. Recombinant adeno‐associated
virus (rAAV) has emerged as the most frequently used vehicle for in vivo GTx
compared with other viral delivery systems such as adenoviral or lentiviral vectors. Based on 136 unique clinical trials posted in the US National Library of
Medicine database (ClinicalTrials.gov) before 26 April 2021, a majority of the
clinical applications fall into the therapeutic areas of ocular diseases, lysosomal
storage disorders (LSD), blood disorders, neuromuscular disorders, and central
nervous disorders[1]. Most of these are inherited rare diseases.
The natural occurring AAVs are small non‐enveloped icosahedral virion of
~26 nm in diameter that contain a single‐stranded DNA genome of 4.7 kilobase
long. Flanked between two inverted terminal repeats (ITRs) are the two major
open reading frames Rep (Replication) and Cap (Capsid). Rep encodes proteins
required for viral replication and packaging, whereas the Cap gene produces structural proteins that encapsulate viral genome and direct the capsid’s binding to
receptors on host cells [1]. Various AAV serotypes that differ in capsid protein
sequences have been isolated from a number of species, such as humans and
Drug Development for Gene Therapy: Translational Biomarkers, Bioanalysis, and Companion
Diagnostics, First Edition. Edited by Yanmei Lu and Boris Gorovits.
© 2024 John Wiley & Sons, Inc. Published 2024 by John Wiley & Sons, Inc.
3 Biomarker and Bioanalytical Readouts for the Development of AAV Gene Therapy
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nonhuman primates (NHP). These diverse serotypes have broad but different tissue tropism profiles determined by their binding to distinct set of receptors on host
cell surfaces. As a result, AAVs can transduce and deliver the DNA payload to a
wide range of mammalian cells and tissues, such as liver, muscle, retina, or brain.
With AAV vector, the coding sequence between the two ITRs is replaced by an
engineered DNA expression cassette to introduce a therapeutic gene of interest (also
known as transgene) to target tissues in humans. ITRs are responsible for transgene
packaging and are the only viral sequences required in rAAV vectors. The technology
platforms for clinical applications can be divided into four different approaches: gene
replacement, gene addition, gene editing, and gene regulation. Corresponding to the
four classes, the transgene may (1) encode a functional gene to replace the patient’s
defective gene; (2) overexpress an endogenous gene or synthetic gene; (3) encode
gene editing components like zinc finger nucleus (ZFN), transcription
activator‐like
effector nuclease (TALEN) or clustered regularly interspaced short palindromic
repeats (CRISPR) associated nuclease 9 (Cas9) to modify the host genome; and
(4) encode gene regulation agents like zinc finger protein‐transcriptional factor (ZFP‐
TF), microRNA or small hairpin RNA to suppress or activate a gene[2].
Unlike most other viruses, AAV is naturally replication‐deficient unless in the
presence of helper virus, such as adenovirus or herpesvirus. The rAAV vectors
lacks Rep and Cap genes and are incapable of replication and packaging in
humans even when coinfected with helper viruses. The viral DNA persists in the
nucleus mostly as an episomal form outside of the host cell’s chromosome. The
circular episomes are stable and provide long‐term gene expression after a singleadministration. The rAAV genome has a low frequency of integration into the
host genome, thus limiting risk of insertional mutagenesis and oncogenicity
[3, 4]. In addition, AAVs are relatively less immunogenic compared with other
viral vectors. These advantageous properties lead to a more favorable risk‐benefit
profile for rAAVs. Glybera (alipogene tiparvovec) was the first licensed product in
Europe in 2012 for familial lipoprotein lipase deficiency. In the United States, the
approval of three AAV GTx products by the Food and Drug Administration (FDA)
has further advanced the field and boosted tremendous interest in this therapeutic
approach: (1) Luxtuna® (voretigene neparvovec‐rzyl) was approved in 2017 for
treating patients greater than 12months of age with inherited retinal disease due
to biallelic RPE65 mutations via targeted delivery to subretina of the eye;
(2) Zolgensma® (onasemnogene abeparvovec‐xioli) was approved in 2019 for
treating patients less than 2 years of age with spinal muscular atrophy (SMA)
caused by biallelic mutations in the survival motor neuron 1 (SMN1) gene via systemic intraventricular (IV) infusion; and (3) Hemgenix® (etranacogene dezaparvovec) was approved in 2022 for the treatment of hemophilia B in adults with
congenital Factor IX deficiency.
For more in‐depth background information, see Chapters 1 and 2.
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3.1.2 Biomarker Category and Utility
The assessment of biomarkers became an undisputable tool in the development of
therapeutics and play a key role in the treatment paradigm change toward precision medicine. A biomarker is not a clinical endpoint that measures how an individual feels, functions, or survives, but rather a defined characteristic that is
measured as an indicator of normal biological processes, pathogenic processes, or
responses to an exposure or intervention, including therapeutic interventions[5].
The type of biomarkers may include molecular, histologic, radiographic, or physiologic characteristics. Biomarker‐driven drug development might have advantages
over a conventional approach, as they have the potential to predict drug efficacy
more quickly than conventional clinical endpoints as well as to accelerate drug
development in certain disease areas. The concept of biomarkers is not new. The
ancient Egyptians used pregnancy tests, a thousand years later Hippocrates noted
the relationship between various visible manifestations of diseases, and finally in
the 1950s the term “biological marker” was introduced followed by widespread use
of “biomarkers” as of the 1980s. In 1998, the first biomarker‐guided drug trastuzumab was approved by the FDA. Finally, in 2015, the FDA–National Institute of
Health (NIH) Joint Leadership Council identified the harmonization of terms used
in translational science and medical product development as a priority need, with
a focus on terms related to study endpoints and biomarkers. Working together with
the goals of improving communication, aligning expectations, and improving
scientific understanding, the two agencies developed the BEST (Biomarkers,
EndpointS, and other Tools) Resource[5]. The first phase of BEST comprises a
glossary that clarifies important definitions and describes some of the hierarchical
relationships, connections, and dependencies among the terms it contains to harmonize and clarify terms of translational science in medical drug development.
The glossary is considered a living document.
According to the BEST glossary, there are seven biomarker categories, namely
diagnostic, monitoring, predictive, prognostic, PD/response, safety, and susceptibility/
risk biomarkers. Examples are given for the context of use for each of the biomarker
categories in Figure3.1.
Pharmacokinetic (PK) (pharmakon “drug” and kinetikos “moving, putting in
motion”) classically describes the absorption, distribution, metabolism, and excretion of a drug over time after administration. For the development of biologics,
there is a clear distinction between PK and biomarker readouts. In the case of
AAV‐based GTx, it is a contentious topic if a particular readout or analyte belongs
to PK or the biomarker category. One reason being that the active drug will be
produced invivo by the patients’ cells, after administration of a vehicle system
(AAV capsid) containing all relevant information (DNA). Generally, the assessment of biodistribution and shedding is seen as the equivalent of PK.
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Diagnostic
Monitoring
Predictive
Prognostic
Biomarker categories
Pharmacodynamic/
Response
Safety
Susceptibility/
Risk
Patient selection
Detect a change in the degree or extent of a disease
Indicate toxicity or assess safety
Provide evidence of exposure
Identify individuals on the basis of effect
from a specic intervention or exposure
Stratify patients
Enrichment: inclusion/exclusion data
Efficacy biomarker/surrogate endpoint
Show biological response related to
an intervention/exposure
Indicate the presence or extent of toxicity
related to an intervention or exposure
Indicate the potential for developing a
disease or sensitivity to an exposure
Context of use examples
Figure3.1 Biomarker categories according to BEST and examples of context of use.
Source: https://www.fda.gov/drugs/biomarker- qualification- program/context- use.
Public domain.
This chapter provides a high‐level overview of the following topics: viral biodistribution and shedding, pharmacodynamic biomarkers of transgene expression
and changes of substrate and downstream pathway markers, assessment of genotoxicity caused by rAAV integration and off‐target gene editing, biomarkers for
immune‐mediated toxicity, safety biomarkers for nonimmune organ‐specific toxicity, predictive and diagnostic biomarkers for study enrollment and patient
stratification.
3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD)
Biomarkers
3.2.1 Viral Biodistribution and Shedding
Though different AAV serotypes have high transduction efficiency toward different set of cell types and tissue, the tropism is not completely restrictive and nontarget tissues may be transduced. Unlike conventional PK studies for biologics
and small molecule drugs, GTx performs nonclinical biodistribution studies to
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evaluate drug exposure and safety[6]. Biodistribution studies in animals, stand‐
alone or more often combined with good laboratory practice (GLP)‐compliant
toxicity study, study the spread of virus within the body, including target and nontarget tissues from the site of administration. Assessments include the kinetics of
vector distribution, persistence, and clearance profile at peak and one (or more)
later time points.
Based on regulatory guidance (US Food and Drug Administration [FDA] guidance), the minimum panel of tissue collection should include liver, gonads, brain,
heart, spleen, kidney, lung, blood, and injection site. Additional tissues may need
to be collected depends on route of administration (ROA), vector type, and mechanism of action[7]. For example, intravenous administration and cerebrospinal
fluid (CSF) delivery of AAV vectors has been shown to cause dorsal root ganglia
(DRG) toxicity in multiple animal species[8–10] and therefore, DRG may need to
be included in biodistribution study for the relevant ROAs. International Council
for Harmonisation guideline S12 recommended a broader panel that includes
adrenal gland and spinal cord collection[11]. In long‐term follow‐up studies in
humans, surrogate samples can be used to test for vector sequence. Chapter4 of
this book has detailed descriptions of the design and conduct of biodistribution
studies and PK and PD evaluations.
The wildly used bioanalytical methods to measure vector genome copy number
from tissue DNA are quantitative polymerase chain reaction (qPCR) (also referred
to as real‐time PCR) and more recently, digital PCR (dPCR). Both methods can
achieve FDA recommended lower limit of quantitation of 50 copies of vector
genome per 1 μg of genomic DNA[12]. dPCR has superior precision than qPCR
but narrower dynamic range and lower throughput.
A shedding study examines the dissemination of viral vectors through secretion
and/or excreta (urine, feces, semen, saliva, etc.) from the patient to assess the risk
of horizontal and environmental transmission. Nonclinical shedding data provide
information on the likelihood of transmission to untreated human individuals
(e.g. caregivers) and inform the design of shedding studies in clinical trial.
Additionally, vector copy numbers in blood circulation must be determined as
part of the PK analysis. The primary assay for shedding analysis is to measure vector genome copy number via qPCR or dPCR which is specific, sensitive, reproducible, high throughput with rapid turnaround. These methods, however, cannot
differentiate intact vs. noninfectious or degraded vector sequences. An infectivity
assay using invitro culture of samples with a permissive cell line followed by
qPCR endpoint analysis can accurately assess the nature of the shed material. For
replication‐competent virus, it is important to characterize infectivity as secondary analysis. For replication‐incompetent virus like rAAV, the infectivity assay
results are informational. The limitations of this type of assay are poor sensitivity
and precision. It is plausible not to perform this test if qPCR/dPCR results are
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below the limit of detection of the infectivity assay[13]. For additional analytical‐
related details, see Chapter7.
3.2.2 Transgene mRNA Expression
The basic elements of a transgene expression cassette typically include a promoter/
enhancer at the 5’ end, gene of interest, and polyadenylation sequence at the
3’ end. Systemic IV delivery results in broad tissue distribution. The majority of the
clinical trials using an IV delivery route for indications such as blood disorders (e.g.
hemophilia A and B) employed tissue‐specific promoters to minimize transgene
expression outside of target tissues, which may help with a better safety profile.
Most of the central nervous system disorders have used more targeted local delivery (e.g. intrathecal and intracerebral) incorporating a ubiquitous promoter[1].
After single‐stranded AAV (ssAAV) genome is taken by a cell, the vector undergoes
uncoating in the nucleus followed by de novo synthesis of complementary DNA
strand to form double‐stranded DNA before mRNA can be expressed. The engineered self‐complementary AAV (scAAV) can bypass the host‐cell DNA synthesis
and immediately reanneal, thereby can produce transgene product much earlier
than ssAAV. The kinetics of vector distribution and transgene expression coupled
with other safety endpoints such as clinical pathology and histopathology can help
determine whether vector presence correlates with adverse findings. Additionally,
characterization of mRNA expression can be used as an efficacy endpoint when
vector‐encoded protein cannot be analytically distinguished from host native protein. High level of transgene mRNA expression may induce stress to the cell
machinery [14] and therefore, in addition to vector genome copy numbers,
transgene mRNA expression in different tissues is also a critical readout for biodistribution and/or GLP‐toxicology studies when evaluating drug exposure and nonclinical safety.
The most frequently used method for mRNA expression is reverse transcription
(RT)‐qPCR. The mRNA is first transcribed into single‐stranded complementary
DNA (cDNA) by reverse transcriptase. The cDNA is subsequently used as the template for DNA polymerase in qPCR amplification or most recently dPCR. In situations to define proportion and/or uniformity of transduction and gene expression
in target cell populations, image analysis of in situ hybridization signals of tissue
sections can be a powerful technique to provide spatial information[6]. These and
additional technologies for mRNA expression analysis can be found in Chapter8.
3.2.3 Transgene and Target Protein Activity and Concentration
Gene therapies address the underlying cause of genetic disorders and is often considered a disease modifying therapy. For gene replacement and gene addition
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applications, the transgene encodes the therapeutic protein directly. For gene editing platforms, the transgene codes for molecule(s) to modify the host genome to
correct mutation(s) in a target gene using technologies such as base editing and
prime editing or insert a functional gene by creating a double‐stranded break in
the target DNA using nuclease of choice. Transgene can also encode proteins
(transcription activator or repressor that are capable of binding to specific DNA
sequences) or RNAs (shRNA or microRNA) to modulate levels of target protein.
All approaches result in production or reduction of a target protein to exert a therapeutic effect. After being taken up by cells via receptor‐mediated endocytosis,
rAAV enters the nucleus and releases the DNA by uncoating the capsid. The
single‐stranded DNA genome undergoes second‐strand synthesis and forms stable, circular, and double‐stranded DNA episome, which provides transgene expression over long term[15].
To achieve the desired level of target proteins, much effort has been invested
into early design of vector constructs. The first step is to choose either naturally
occurring capsid types with high tropism toward target tissue or engineered novel
capsids with improved transduction efficiency and selectivity for target tissue(s).
Additionally, engineering DNA expression cassette by incorporating a strong promoter (ubiquitous or tissue‐specific) and/or RNA stabilization motif (such as
mutated Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) is a
second approach to reach high level of transgene expression.
Transgene or target protein expression is an essential readout in preclinical and
clinical pharmacological assessment because the expression can correct the molecular defects of the diseases. Proof‐of‐concept studies in animal disease models are
important to demonstrate that a certain level of gene or protein expression can
positively impact disease. Duration of the expression is another important characteristic of a successful treatment. Factor IX expression in human muscle tissue has
been shown to last up to 10 years post a single administration[16]. However, loss
of transgene expression of factor VIII and IX have been reported in humans[17, 18].
Multiple factors can affect the durability of transgene expression. The host cell‐
intrinsic innate immune system can detect the capsid and DNA genomes rich in
hypomethylated 5’‐cytosine‐phosphate‐guanine‐3’ (CpG) dinucleotides through
pattern recognition receptors such as Toll‐like receptors (TLRs) 2 and 9, respectively. The TLRs activate the myeloid differentiation factor 88 (MyD88) pathway
leading to proinflammatory type 1interferon response and augmented adaptive
immune response to capsid and/or the transgene product, which may result in
destruction of transduced cells and loss of transgene expression[18]. Detectable
capsid‐specific cytotoxic T lymphocytes in the circulation have been linked to liver
enzyme elevation in several clinical trials. Some of the subjects have lost transgene
expression likely due to CD8+ T‐cell killing of transduced cells that display capsid
antigen. There are also non‐immunological factors that may affect the durability of
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transgene expression. Terminal differentiated (neurons) or long‐lived cells (muscle
myocytes and hepatocytes) can maintain circular episomes for long term, whereas
rapid dividing cells may dilute the episomes through rounds of replication[19, 20].
Lastly, cellular stress from transgene overexpression may trigger apoptotic signaling leading to death of transduced cells[15].
Most inherited metabolic disorders such as LSDs are caused by “loss‐of‐
genetic mutations in genes that encode enzymes. For GTx that corrects the enzyme
deficiency, enzyme activity measurement is preferred over protein quantitation as this
is a direct readout of the biological activity of the enzyme that indicates efficacy. For
blood disorders such as hemophilia, coagulation activity of the transgene product is
also preferred over protein levels. These activity readouts can serve as a surrogate endpoint for efficacy. For transgene and target gene products that are proteins with no
activity, the amount of protein in biofluid or tissue biopsies can be measured by ligand
binding assay or other technologies, e.g. immuno‐affinity mass spectrometry, immunohistochemistry, and Western blotting or similar techniques[21]. The activity and
protein expression of transgene or target gene products, together with PD biomarker
readouts are critical parameters for PK/PD modeling and clinical dose selection.
GTx transgene products are expressed endogenously from the target cells. This
is different from small molecules and biologics which exert therapeutic effects
directly after administration without the need of further in vivo processing.
Enzyme activity determination and protein quantitation for endogenous analytes
pose bioanalytical challenges. Chapter9 describes solutions to overcome various
unique challenges.
function”
3.2.4 Substrate and Other Distal PD Biomarkers
Genetic mutations of inherited metabolic disorders often affect enzymes in metabolic pathways. They are typically grouped by the affected substrates such as
amino acids, carbohydrates, lipids, purines, and pyrimidines. A large class of
inherited metabolic disorders are LSDs caused by deficiency in lysosomal enzymes
of acidic hydrolases that are capable of digesting macromolecules such as glycoproteins and lipids. The absence of the functional enzyme typically results in toxic
substrate buildup and/or loss of essential metabolites that cause organ damage[22]. Some of the other inherited metabolic disorders are phenylketonuria
where deficiency of phenylalanine hydroxylase results in high levels of phenylalanine in the blood; glycogen storage diseases with mutant enzymes that affect
glycogen metabolism, and peroxisomal disorders with defective enzymes resided
in peroxisomes. In vivo production of a functional protein through GTx would
potentially clear the substrate buildup and restore metabolites.
Substrates, metabolites, and other molecules in the downstream pathway are
primary disease biomarkers that reflect disease pathogenesis, making them ideal
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