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1 Introduction to AAV-based invivo Gene Therapy
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in diagnostics, safety, response, and monitoring. A more detailed discussion can
be found in Chapter3.
Translational biomarkers support the translation from data obtained in preclinical research to clinical trials. These assessments address the sensitivity, specificity, and toxicity of the AAV‐based gene therapy and inform measures of therapeutic
efficacy and prognosis. Structural biomarkers assess tissues and organs through
imaging methodologies, such as ultrasound, MRI, and PET. Functional biomarkers can assess clinical endpoints related to organ functionality and prognosis,
such as motor and cognitive clinical assessments in various CNSCNS and muscular degenerative diseases. Liver and kidney function biomarkers can be assessed
in the blood to measure their function and monitor treatment responses of liver
and kidney‐targeted gene therapies. Preexisting antibodies to AAV can be used as
biomarkers for the prediction of AAV gene therapy effectiveness and often need to
be developed into companion diagnostics. AAV antibodies present may be a predictor for AAV gene therapy response and can indicate a need for adjuncts to
prevent the rejection of transfected cells. Tumorigenesis biomarkers can assess
the potential development of neoplasia. Tumor testing and profiling biomarkers
can help identify the type of tumor and oncogenesis. Genetic testing can determine a person’s risk of certain cancers or the type of cancer‐causing tumor.
Genetic testing, specifically of a tumor, can give further information about its
growth and other characteristics.
1.5.1 Gene Therapy Clinical Trials: Spotlight onHemophilia A
As a representative example of the clinical development processes for gene therapies, we will reference the trial design for patients with severe or moderately
severe forms of hemophilia A. This has been extensively reviewed by Pipe etal.,
showing that, in contrast to several biopharmaceutical companies developing
gene replacement therapies based on the B‐domain deleted human factor VIII,
ASC therapeutics is introducing a second‐generation gene therapy based on a
B‐domain deleted human/porcine chimeric factor VIII[79]. This next‐generation
gene therapy has shown in preclinical studies the potential to decrease dose
requirements, increase durability through improved factor VIII expression, and
improve durability driven by a more efficient expression and reduced intracellular stress.
A first‐in‐human, open‐label, dose‐finding study is designed to assess the safety
and preliminary efficacy of a single infusion of the AAV8‐based chimeric
transgene replacing the coagulation protein factor VIII in study participants with
severe and moderately severe hemophilia A (FVIII activity ≤ 2 International
Units/Deciliter).
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 21
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In this study, the main safety monitoring parameters include:
1) Physical examination, including assessments of general appearance; head,
eyes, ears, nose, and throat; the cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, genitourinary, musculoskeletal, and neurologic systems. Height and weight will also be measured and recorded.
2) Vital signs, including oral temperature, pulse rate, respiratory rate, and blood
pressure.
3) Viral shedding, assessing the evidence of potential viral transmission, will be
tested on samples of blood, saliva, urine, stool, and semen.
4) Liver ultrasound.
The main efficacy monitoring assessments include:
1) FVIII activity, determined by validated assays, one‐stage activated partial throm-
boplastin time (aPTT), and chromogenic FXa. FVIII levels should be taken at a
trough or close to trough levels, meaning after a minimum of 72 hours has
elapsed since the last infusion of FVIII protein concentrates.
2) Bleeding episodes and FVIII replacement therapy will be captured in the par-
ticipant’s diary. In addition, the number of bleeding episodes requiring treatment, and the number of bleedings that do not require treatment following the
administration of ASC618infusion, will be recorded throughout the study.
The main exploratory assessments include:
1) Hemophilia A quality of life (Haem‐A‐QoL) questionnaire was completed by
participants during the study.
2) Blood samples will be collected for potential future research in the hemophilia
A disease area to assess: (1) Liver structure, including Fibroscan and abdominal ultrasound; (2) Liver function, including liver enzymes, GRP78/BiP, and
microRNA exploration analysis; (3) Liver tumorigenesis, including GALAD
score (serum biomarker‐based model that predicts the probability of having
HCC*, incorporating gender, age, AFP‐L3, AFP, and DCP), VirScan (comprehensively analyzing antiviral antibodies), and cell‐free DNA assessment.
To complete these assessments, extensive monitoring, including on‐site and
remote visits, will be implemented. The levels of FVIII and liver function will be
evaluated twice a week in the first 16weeks, slowly decreasing as these measurements reach a plateau.
It is crucial during gene therapy to have measures of improvement, disease
stage, or to prescreen for predicting its effectiveness and any need for adjunct
therapy, for example, to suppress an immune reaction that may reject the
AAV‐infected cells (NCT04676048).
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1.6 Predictions forScientific and Medical Progress
Over the past decade, approximately 45% of gene therapy clinical trials have
focused on invivo as opposed to exvivo gene therapy[76]. Many platforms are used
for AAV‐based gene therapy, including cDNA gene replacement, gene editing,
genome regulation, and gene addition. The development and use of translational
biomarkers are incredibly important for predictive and diagnostic measures during
gene therapy clinical trials. A critical step in preclinical testing is a thorough analysis of off‐target effects. As mentioned earlier, analysis software systems can aid in
the detection and monitoring of off‐target effects. Antibodies against the capsid,
protein expression and activity levels, and viral shedding are just a few examples of
measures used as predictive, diagnostic, and safety biomarkers. Immunogenicity is
another important factor that should be monitored via biomarkers.
Biodistribution and shedding are typically analyzed through polymerase chain
reaction (PCR), while transgene mRNA expression analysis can be performed
using reverse transcription‐PCR and in situ hybridization (ISH). Transgene protein concentration can be measured by numerous platforms – enzyme‐linked
immunosorbent assay (ELISA), electrochemiluminescence (ECL), immunoPCR,
liquid chromatography–mass spectrometry (LC–MS), Western blot, and high‐
performance liquid chromatography (HPLC). Immune response measurements
can be performed using ELISPOT, flow cytometry, electrochemiluminescence,
and more. Genome integration must also be measured after treatment, which
helps discern potential carcinogenesis. More safety, efficacy, and diagnostic biomarkers, companion diagnostics, and gene therapy treatments are sure to emerge
continuously. Regulatory practices are essential in ensuring the study is worth the
effort and the subjects’ rights are protected. In addition, guidance surrounding
thedevelopment of gene therapies, useful biomarkers, and companion diagnostics needs to be expanded.
1.6.1 Predictions forChallenges inthe Field
Among the major factors preventing AAV‐driven gene therapies from being more
broadly adopted are challenges related to scalability, such as large‐scale production and cost‐effectiveness. Manufacturing gene therapies typically begins with
plasmid development and production, then cell expansion, plasmid transfection,
viral vector production, purification, and finally, formulation. Companies often
use third parties to complete this process; however, competition for these services
with other advanced therapies, such as mRNA vaccine development for COVID‐19,
may become a significant bottleneck. Thankfully, the development of the Sf9 system has been integral in making large‐scale manufacturing more possible by
decreasing the amount of viral particles needed for efficient production[30].
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Thisincreased demand for third‐party manufacturing services drives many companies to establish in‐house manufacturing.
Several challenges are related to ensuring a balanced risk‐benefit solution, in
other words, achieving a safe and therapeutic gene therapy. Large doses may be
necessary to reach a desired therapeutic effect, increasing the likelihood of side
effects and costs. Thus, the lowest effective dose should be determined. Transgene
expression needs to be specific to the target cell or tissue to minimize off‐target
effects and be long‐lasting. We are likely to see more CRISPR use in the future of
in vivo gene therapy, but for now, off‐target effects are the main concern. The
immune system needs to stay unresponsive to the viral vector in order to maximize efficacy, enable redosing, and prevent toxicity. Many strategies have been
and are being developed to help AAV‐based therapies evade the immune system,
including codon optimization, CpG reduction, regulatory elements, integrative
AAV vector approaches, lower dosing, and immunomodulation.
To overcome these challenges, it is vital to identify translational biomarkers to
measure a particular therapy’s durability, immunogenicity, malignancy, and toxicity. Recent adverse events and deaths have led to clinical holds on some gene
therapies. Thus, the FDA has closely monitored gene therapy safety in the United
States and the European Medicines Agency (EMA) in the EU.
Translational biomarkers, either primary, secondary, or exploratory, i.e. surrogate endpoints include efficacy biomarkers, defining the clinical activity of the
gene replacement therapy (i.e. durability can be measured through the length of
effectiveness observed by normal blood levels, staying in remission, or physical
symptom abatement); safety biomarkers to assess dose‐limiting toxicity; tumorigenic biomarkers to assess malignancy risk of organs targeted as biofactories
for gene therapy; immunogenicity biomarkers, which can be used as inclusion/
exclusion criteria, especially anti‐AAV preexisting antibodies produced by previous exposure to an AAV. These antibodies can significantly impact the effectiveness
and eligibility for a particular treatment.
Recent focus has been placed on developing safe strategies to block anti‐AAV
antibody effects. Among the alternatives to overcome these challenges are isolated liver perfusion, which can be effective but may induce inflammation; reduction of IgG levels, a method undergoing preclinical trials of autoimmunity;
plasmapheresis, a potential approach that may require several therapeutic rounds
for effective depletion of anti‐AAV neutralizing antibodies; and capsid modifications as a method to prevent neutralization.
1.6.2 Addressing Durability
AAVs have the potential for long‐lasting effects, which is one of the main goals of
gene therapies. Gene therapy seeks to provide a onetime treatment for patients.
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This is incredibly important for those who currently have inadequate treatment
options that may have rigorous regimens or substantial side effects and who have
no available treatments on the market. The immune response considerably influences durability and efficiency, as existing antibodies may activate immune cells
that prevent viral transduction.
1.6.3 Addressing Immunogenicity
The percentage of people possessing preexisting anti‐AAV antibodies is quite significant. It has been estimated that anti‐AAV1 and anti‐AAV2 antibodies can be
found in 70% of the population[80]. Anti‐AAV6, 8, and 9 antibodies have a lower
incidence of approximately 30–50%, while the overall incidence of all serotypes is
estimated at 30–70%. These rates are impacted by geographical location and
increase with age[80]. A person’s level of anti‐AAV antibodies, typically measured by ligand binding assay, can be used as a biomarker for inclusion/exclusion
criteria. Measurements can be performed using cell‐based transduction inhibition
assay for neutralizing activity as well. Thankfully, several strategies have been discussed and suggested for addressing immunogenic responses to gene therapies.
There is potential for an immune response to the capsid or the transgene itself
posttreatment. Capsid modifications have been suggested as a potential method to
prevent neutralization by antibodies[81]. Immunosuppressive drugs show promise for pretreatment in seropositive patients, but more testing is necessary. Strong
promotors are vital to lowering the dose necessary for a therapeutic effect and,
thus, reduce the potential for adverse effects[82]. Further detail of immunogenicity will be discussed in Chapter5.
1.6.4 Addressing Malignancy
AAV‐based gene therapies have shown less malignancy than the use of other vectors; however, recent research has shown more risks than were thought to exist
prior. The development of sensitive tumorigenic markers is essential for understanding the risk of malignancy during development and clinical trials. As
research advances and continual developments are made, gene therapy will
become more targeted, effective, and safe.
1.7 Predictions forMarket Adoption
With the rise in chronic diseases such as cancers, neurodegenerative diseases,
genetic disorders, and rare diseases, the market for gene therapy has grown
greatly. The most commonly targeted tissues of invivo gene therapy clinical
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trials over the past decade have been the CNS, heart, liver, eye, bone marrow,
and tumors[83]. Pharmaceutical and biotech companies have been the biggest
contributors to the gene therapy market. Some large competitors in the gene
therapy market include Novartis, Sangamo Therapeutics, and Biogen to name a
few, although many partnerships seem to be transpiring between these companies to produce invivo therapies[84]. The Q2 report of 2022 released by the
American Society of Gene & Cell Therapy (ASGCT) and PharmaIntelligence
reports that 2024 gene therapies were under development and testing in the
preclinical, clinical trial, or preregistration phases. Several market assessment
reports have estimated that the market for gene therapy will grow at a 20–30%
annual rate throughout 2030.
1.7.1 Patients and Patient Advocacy Groups
Several gene therapy patient advocacy groups help advance research, educate people about gene therapy, and help with treatment costs. ASGCT is a professional
organization made up of scientists, physicians, and patient advocates with the
goal of enhancing knowledge, education, and awareness about gene and cell therapy. Gene and cell therapy clinical trials and landscape reports can be found on
their website. Askbio is a similar site with a collaborative program called AskFirst,
where patients and their families are “asked” how they can help. Askbio also has
a list of advocacy partners that help with education, research, and events to benefit patients. To name a few, CureDuchenne, Speak Foundation, Angelman
Syndrome Foundation, Defeat MSA Alliance, and Nationwide Children’s Hospital.
1.7.2 Physicians, Clinical Guidelines, Regulatory Agencies
Regulatory approval for gene therapy involves several regulatory boards that vary
by country. The EU and United States have different oversight in that the FDA
oversees all US clinical trials and follows a drug’s lifecycle from its conception. In
contrast, the EMA is a reviewing body over many European nations. Each state
has its own regulatory body that follows the clinical trial. New drug applications
in the United States go through the FDA, and in the EU, they can either go through
a state’s regulatory committee or the EMA. The time it takes drugs to be approved
is a long process, and the FDA’s reviewing process has been thought to be much
longer; however, a study published in 2011 found that the process through the
EMA is longer. When looking at approval for cancer drugs, they found that new
drugs were reaching patients in the United States faster than in Europe due to a
faster review process by the FDA[85].
Before clinical trials begin in the United States, an investigational new drug
application (IND) must be submitted to the FDA and approved. The Institutional
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Review Board (IRB) reviews and monitors the clinical research to ensure compliance throughout a trial. The trial protocol, consent form, HIPPA authorization,
etc., must be presented to the IRB for review and approval. Study modifications
and amendments, adverse events, and annual reviews are required by the IRB to
be submitted and approved promptly to stay compliant and complete the trial.
More information will be presented in Chapter18.
1.7.3 Payers
Most gene therapies are likely to be covered by insurance in the future, but currently, most policies have yet to cover them. Advocacy groups have been tremendously important for many who needed gene therapy but could not afford the
high costs out of pocket. Experimental treatments are typically free options but
put patients at unknown risks. Clinicaltrials.gov can provide a list of AAV invivo
gene therapies being tested in active clinical trials.
1.8 Final Thoughts
Inherited monogenic rare diseases are ideal targets for gene therapies based on
the success of gene replacement therapies in adults and children; however, gene
therapies are quickly emerging for various non‐monogenic diseases as well.
Preclinical studies have been using various gene editing platforms to correct a
defective gene or insert a functional gene in animal models and/or clinical studies. Sustained expression of gene therapies in humans necessitates lengthy, thorough studies to measure long‐term efficacy and off‐target activity to provide a
positive safety profile. Improved methods in genome editing have dramatically
reduced off‐target and deleterious effects successfully in preclinical models,
needing translation to clinical programs. AAV vectors are currently being utilized by numerous clinical trials to treat numerous, various diseases, and gene
editing will pave the way for achieving successful results with high specificity,
efficacy, and safety.
1.8.1 Can We Afford invivo Gene Therapies?
Large‐scale manufacturing, availability of financial resources, and limited access
to experts are causing the price of gene therapies to skyrocket. The current cost of
Glybera is approximately 1.2million and Luxturna, released in 2017, costs approximately $425,000 per eye. For many researchers, the development cost throughout
the preclinical to clinical phase is not feasible. The need for toxicology, safety, and
dosing studies and IND approval creates more hurdles with approval and cost.
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Inherited monogenic rare disease treatment is costly and continual, with enormous financial burdens. Advancing therapies for restoring defective protein levels
have a tremendous impact on people suffering from rare and currently untreatable diseases. Hopefully, with the development of advanced technologies, the creation of cheaper, safe, and successful methods will lower the costs for both
production and patient treatment. This is promising as we see the development of
novel tools for measuring, monitoring, and preventing adverse effects and the success of gene therapies.
1.8.2 Can invivo Gene Editing Replace Gene Therapy?
While incredible progress in gene editing has been made in the past 15 years,
there are many areas that still need substantial improvement. Key elements
required for transitioning from gene therapy to gene editing include overcoming
significant challenges. New therapies need to concentrate on overcoming transient expression, reducing off‐target activity, and lowering the risk of genotoxicity
following long‐term exposure. Efforts should be made to minimize the need for
repeated injections of gene therapies, which are more likely to trigger an immune
response, lowering the efficacy while reducing its safety.
Delivery of gene‐editing techniques has been achieved with both viral and non‐
viral delivery systems. Viral delivery includes AAV vectors while alternative vehicles like lipid nanoparticles can easily pass through cell membranes and have seen
increased use in targeting the liver without activation of the immune system,
allowing for repeated administration. Additionally, magnetic nanoparticles have
successfully delivered gene‐editing systems with little off‐target effects and no
toxicity [86, 87]. A nuclease‐free system can be paired with CRISPR/SaCas9,
which has shown significant improvements in targeting effectiveness and stable
gene modification in mice[88]. Studies utilizing these newer technologies are still
in their infancy for invivo applications.
Unfortunately, the major barrier to both gene therapy and gene editing is the
immune response. However, patients can be given prophylactic steroid treatments, prior, to reduce the immune response[89]. Another major concern is long‐
term stability. While AAV vectors are technically capable of cargo delivery to
mitotic and postmitotic cells, in practice, the delivery is typically episomal, resulting in diminished effects as cells divide. This is particularly relevant in cases
involving pediatric patients, where attempts to cure monogenic liver diseases
have been met with concerns about hepatocyte proliferation[90–92]. As discussed
previously, repeated injections of AAV vectors lead to the production of anti‐AAV
neutralizing antibodies, reducing efficacy and safety of subsequent treatments or
therapies[40, 93–95]. In patients who have undergone AAV therapy and developed anti‐AAV antibodies, utilizing non‐viral delivery of CRISPR/Cas9would be
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optimal as it would not be affected by anti‐AAV antibodies. In summary, invivo
gene therapies have limitations that can be overcome with invivo gene editing,
but significant developments and research are needed before the safety and efficacy of treatments are shown and optimized.
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