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9
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Quantification ofTransgene Protein Expression and
Biochemical Function
Robert Dodge1 and Liching Cao
1
Department of BioMedical Research, Novartis, East Hanover, NJ, USA
2
Biomarker and BioAnalytical Sciences, Sangamo Therapeutics, Richmond, CA, USA
2
9.1 Introduction
Many transgene proteins in gene replacement therapy are from protein classes not
typically encountered in traditional bioanalysis of soluble biomarkers or targets of
biotherapeutics. Those classes include intracellular proteins located in subcellular
compartments or structural proteins, or membrane proteins which require new
methods and platforms to be developed to high quantitative standard in addition
to traditional methods like EnzymeLinked Immunosorbent Assays (ELISA). This
chapter aims to discuss the current industrial practices and challenges encountered during method development and qualification/validation of transgene protein quantitation in terms of expression and biochemical function.
Discovery and early development gene therapy studies are carried out in animals. Because of that, there are many more tools available for obtaining samples
and assessing protein expression and biodistribution that may not be available in
the clinical setting. Most notably, assessing expression in a variety of tissues may
only be possible in animals except in the very limited cases where planning and
consent are available to test human tissue for transgene expression retrieved during a human autopsy[1].
Because of the challenges with quantification of transgene protein expression
as presented in this chapter, most gene therapy biodistribution studies are carried
out by assessing transgene DNA levels, or sometimes transgene mRNA. However,
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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.
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the ability to test protein expression in animal models has some advantages. For
example, detecting transgene DNA in the brain may be useful and predictive; however, proof of expression of a functional transgene protein may be valuable and
more conclusive of effective entry of a functional transgene DNA into cells. And
while the presence of transgenetranslated mRNA in a cell or tissue is generally
predictive of transgene protein expression, determination of functional transgene
protein will always be the most effective pharmacodynamic marker indicating success of a gene therapy treatment.
9.2 Transgene Protein Concentration Determination
9.2.1 Human Transgene inPreclinical Species
As many gene therapy transgene proteins are wildtype proteins, designed to be a
correction for genetic mutations that exist in the disease state, or cause the
increased expression of an already endogenous expressed protein, the ability to
distinguish the transgene protein of interest over the background of existing
protein in the animal. In animal models, this may be especially true of a human
transgene in the gene therapy therapeutic has considerable homology to the animal protein. This creates a challenge for the bioanalytically determination of
transgene protein expression measurements[2]. In addition, as most transgene
protein bioanalytical work is done in animal models, where functional and
homologous transgene protein is likely expressed absent a knockout or genetically
modified animal and so differentiating the human gene therapy transgene product from the endogenous animal protein is challenging. Typically, the ability to
detect small variations in two otherwise equivalent proteins, with specific antibodies in a ligand binding assay (LBA) is challenging. While it is possible to generate an antibody that would distinguish, for example, between a genetically
mutated protein and transgene with the wildtype sequence, access to these specific antibodies or generating these antibodies de novo may not be possible within
timeframe needed for testing. Hence, the major bioanalytical methodology for
assessing expression of a transgene when an endogenous or very similar animal
protein exists is mass spectrometry (MS) assay protein formats as discussed further in the chapter. With MS, the ability to distinguish differences between two
similar proteins differing only slightly in amino acid sequence and thus mass is
possible and straightforward. Because of this, most transgene protein assessments
of human gene therapy therapeutics are carried out with MS platforms.
9.2.2 Human Transgene Assessment forIntracellular Proteins
While most early development and discovery studies are done in animals or even
specific animal models, ultimately the most interesting and scientifically relevant
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pharmacodynamic measurements are in humans. As described in this chapter,
measurement of gene therapy therapeutic transgene proteins is highly dependent
on the type of transgene product, from bioanalytical assay technique to limitations on obtaining relevant samples.
Measurement of intracellular proteins can be illustrated by the case study with
the assessment of the intracellular transgene survival motor neuron (SMN) protein
in the gene therapy therapeutic ZOLGENSMA® (onasemnogene abeparvovecxioi).
This work and strategies for transgene assessment provides a comprehensive
understanding of transgene protein assessment for intracellular proteins.
Spinal muscular atrophy (SMA) is a neuromuscular disease that affects the part
of the nervous system that controls voluntary muscle movement. Unlike many
other neuromuscular diseases, there is a clear understanding of the specific genetic
cause of SMA and that results directly from biallelic mutations in the survival
motor neuron 1 (SMN1) gene. Therefore, direct measurement and
concentration levels in various solid and liquid tissues of SMN transgene protein
that Zolgensma gene therapy provides are of scientific interest.
Development of an electrochemiluminescence (ECL) immunoassay for detection and quantification of SMN protein has been reported using standard sandwich immunoassay format with antiSMN antibodies used for capture and
detection reagents[3]. As will be relevant for any transgene protein expressed,
stability exvivo will be important to assess to ensure accurate interpretation of
results. This is important because the environment of a sample exvivo will be very
different than the intracellular environment. In addition, many intracellular proteins may not have a long halflife, and so maintaining the protein exvivo in stable
form prior to measurements of functional assessments can be challenging. In the
work of Zaworski etal., stability was carefully assessed and it was noted that room
temperature, 4 and −20°C even was noted to be limited in whole blood. These
challenges with exvivo stability are not unusual in a protein expressed invivo
intracellularly, especially in this case where measurements were made in blood,
which would not be the native environment for this transgene protein and thus
stability in storage in blood may be expected to be limited. Although careful fractionation of whole blood showed that red cell or platelet levels of SMN were the
primary source of SMN protein in whole blood.
Once a sensitive and robust assay, validated for stability, specificity, parallelism,
and accuracy is available, investigation on correlation between accessible matrix
(whole blood in this case) and disease state or genotype may be carried out. If
there is such a correlation, there may be the ability for transgene protein expression levels to be evaluated in standard matrix like whole blood if the protein is
soluble and functional even if the native environment is primarily intracellular. In
the case of SMA, it was determined that there was a correlation between disease
severity, genotype, and levels of circulating SMN protein in whole blood[4]. Other
work also showed, in general, correlation between mRNA levels and protein
relevant
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levels for most patients, but not all types of patients when analyzed based on time
of disease onset[5].
In addition, use of the SMN protein immunoassay to assess SMN protein levels
after treatment with Zolgensma did not show an obvious increase in the levels of
SMN protein in whole blood. The correlation between mRNA levels, measured
transgene protein concentrations and response to gene therapy treatment may not
be straightforward. Therefore, even when measurements of transgene intracellular proteins are available, it is necessary to examine all data from DNA, mRNA,
and protein levels assays as well as efficacy levels before using transgene protein
expression data for a conclusion.
9.2.3 Human Transgene Protein Assessment
forNon-secreted Proteins
Perhaps the largest area of interest and most developed field of nonsecreted gene
therapy transgene proteins are in ocular gene therapy products[6]. The approval
of LUXTURNA™ (voretigene neparvovecrzyl) in 2017 is evidence of the viability
of this type of therapeutic. Luxterna treats a specific disorder in a class of genetic
hereditary retinal dystrophies that are associated with progressive visual dysfunction and due to mutations in any one of more than 220 different genes. Specially,
Luxterna delivers a gene that codes for the enzyme RPE65. While RPE65 is not
specifically a transmembrane protein, it is expressed in the retina pigment epithelium and is not freely available to measure the expression in the circulating blood.
Because of the challenge of measuring the RPE65 transgeneexpressed product,
all clinical pharmacokinetic results focused only on the measurement of the vector gene using PCR[7].
Even in cell lines or animal expression studies, because of the challenge of
detecting a membraneassociated protein, most studies of expression of RPE65
are inferred by levels of mRNA present as opposed to actual detection of protein
transgene[8]. Thus, detection and quantification of the RPE65 protein has not
been done using traditional bioanalytical methodologies associated with soluble
transgene expression. However, direct detection of the membraneassociated
enzyme RPE65 can be carried out in a semiquantitative manner. For example,
RPE65 protein can be detected from harvested mouse retinas using Western
blots [9]. In this work, retinas were homogenized with detergent and extracts
were electrophoresed on SDSPAGE gels, transferred to nitrocellulose and detected
with antiRPE65 antibodies. Relative levels of protein were determined based on
the relative intensity of the bands on the Western blot relative.
Assessing levels of protein expressed using Western blot imagining and relative
interest can be a very useful technique for assessing membraneassociated proteins
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in animal studies, but is it subject to several caveats. First, while semiquantitative
Western blot analysis within a study or experiment can be very informative, due to
lack of universal standards and potential technique variation between labs, comparison of expression levels in different studies, or between laboratories is challenging. Thus, Western blot quantification is best suited to a specific study and
comparison of conditions rather than universal quantification of transgene protein
levels. Second, Western blot analysis relies extensively on having specific antibodies to the gene of interest. While for RPE65, because of the overall specific interest
and disease state, there are sufficient useful antibodies commercially available, it
may not always be the case for all membraneassociated gene therapy expressed
transgene proteins.
Related to the antibody specificity, as in the case with Luxterna, which is a treatment of patients with confirmed biallelic RPE65mutationassociated retinal dystrophy, the mutated RPE65 protein is present in the disease state. And the antibodies
used in Western blot imaging will likely not be available that are specific enough to
mutated epitope so that they can distinguish between mutated nonfunctional
RPE65 protein and the wildtype functional RPE65 enzyme. Thus, the Western blot
method of quantification of transgene protein products may be of limited utility in
assessing expression levels of transgene after treatment even in animal models.
Relative expression levels can also be determined with immunohistochemistry
(IHC) staining[10]. Relevant sections of the eye can be isolated and fixed using
standard IHC techniques, and then stained with antiRPE65 antibody and detected
with secondary labeled antibody. As in Western blot analysis, relative quantitation
of a membraneassociated protein is primarily semiquantitative and also subject
to the caveats of antibody availability, specificity to mutated vs. wildtype RPE65
protein and variation between experiments and between laboratories. However, an
advantage of the IHC methodology may enable investigation into expression
patterns within tissue at a higher resolution than the Western blot, which generally
involves homogenization of bulk tissue, resulting in loss of specific location expression information.
For the case of RPE65, which is a membraneassociated enzyme, as in secreted
enzymes, it may still be possible to assess enzyme activity. As in the case of
Western blot and IHC methodologies, assessing differences between nonfunctional mutated RPE65in the disease state and functional RPE65, resulting from
transgene expression after gene therapy treatment is not generally possible.
However, enzyme activity assays have been carried out with RPE65 expressed in
the eye[9]. The substrate for RPE65 enzyme has been identified as retinyl esters
and invitro methodologies have been developed to measure RPE65 enzyme activity and have been established and shown in RPE65 enzyme knockout mice[11].
While the extractionhomogenization procedure for all enzymes may not be
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amenable to retaining enzyme activity, RPE65 enzyme activity is possible to be
measured after extraction. For RPE65 transgene expression evaluation, the ability
to distinguish between no or low levels of enzyme activity and increased activity
in the presence of functional RPE65 is clear. As in Western blot and IHC,
between experiments and between labs makes this method semiquantitative.
However, the key advantage of direct detection of enzyme activity allows one to
access the general success of functional protein expression in animals for an
enzyme transgene product delivered using gene therapy techniques.
9.2.4 Human Transgene Protein Assessment forSecreted Proteins
Assessment of protein levels for intracellular proteins or membranebound or
associated proteins has been discussed. Primarily, assessment of proteins in these
cases in human studies may be semiquantitative, or for example, in the case of
SMN protein, measured in whole bold as a potential surrogate to intracellular
concentrations in applicable cell types. In addition, nonsecreted protein concentration or location assessment may be applicable in early discovering animal work
for biodistribution and potentially assisting benefit if an animal model is available.
For secreted proteins, that is those proteins we expect to exist extracellularly,
for example, in blood, the clinical applications for determining protein concentration levels may be much greater. The largest class of secreted proteins, those naturally or expected to be present in the blood, are enzymes and are the most common
gene therapy therapeutics in development. And of the enzyme gene therapy disease targets, hemophilia is highly targeted[12]. Hemophilia is a bleeding disorder
in which the blood does not properly clot. This cause of the disease is primarily
genetic, leading to low levels of Factor VIII or Factor IX. The levels of clotting factor present are typically directly correlated to severity of the disease. Therefore, for
gene therapy therapeutics that are designed to increase levels of Factor VIII or
Factor IX, quantitation of the amount of protein present during treatment will be
a valuable pharmacodynamic marker. Hence, for these secreted transgenes, measurement either concentration or as more typically by enzyme activity is an important pharmacodynamic biomarker.
EtranaDez (AMT061) is a gene therapy using AAV5 to deliver an enhanced functionality Factor IX gene[13] to liver cells. Because of the enhanced functionality of
the transgene and direct correlation of enzyme activity levels to disease severity,
measurement of transgene enzyme activity is a key aspect of clinical development.
In clinical studies, the Factor IX activity levels is a secondary outcome of clinical
trial after the primary outcome, which is a decrease in annualized bleeding rate
(Clinicaltrails.gov NCT03569891,[14]). As described in detail later in this chapter,
enzyme activity assays can be quantitative and provide direct levels of functional
protein levels and thus can be used to assess clinical pharmacodynamics.
variability
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9.2.5 Human Transgene Protein Assessment
forExpressed Therapeutics
The gene therapy transgene proteins discussed so far have been endogenous or
naturally occurring proteins that the gene therapy therapeutic was designed to
provide or enhance levels of. Another class of gene therapy transgene product is a
designed therapeutic that is not a naturally occurring protein. This type of gene
therapy therapeutic would potentially replace repeated doses of a therapeutic protein and instead, have patient’s cells express the therapeutic invivo. The utility of
understanding the concentration of the transgene protein invivo in this class of
gene therapy transgenes is clear because the pharmacokinetics of the original
protein therapeutics delivered with traditional methodologies (e.g. Injections at
intervals) will be well understood and a therapeutic window (safety and efficacy)
based on protein therapeutic concentrations over time will have been established.
Therefore, the ability to measure gene therapyexpressed transgene protein concentrations to verify similar expression levels will be especially important for this
class of gene therapy therapeutics.
RGX314 is an AAV8 vector designed to deliver the transgene for a soluble anti
VEGF Fab and provide continuous antiVEGF therapy. Approved antiVEGF
therapies to treat eye diseases, such as diabetic macular edema, myopic degeneration, and wet agerelated macular degeneration, for example, Avastin, Lucentis,
Eylea. RGX314 delivers the gene sequence to produce the transgene protein with
the sequence of Eylea. The pharmacokinetics of Eylea are described in the drug
label[15] as well as in the literature[16]. Standard bioanalytical assays either validated or qualified can be used to quantitate the transgene protein therapeutic in
plasma as well as other matrices that is possible to collect in human clinical trials,
such as aqueous humor. The phase 2 clinical trial for RGX314includes a secondary outcome for the concentration of the RGX314 transgene product in serum
and aqueous humor (Clinicaltriais.gov NCT04514653,[17]).
9.2.6 Transgene Protein Assay Format Considerations
Prior to initiation of method development for an assay to quantitate transgene protein from gene therapy, several factors should be considered. The most important
consideration will be the type of protein. For example, is the protein an enzyme,
membranebound or membraneassociated, or secreted and expected to circulate
in blood extracellularly. Choosing the platform will depend on those factors as
well as availability of matrix. So an understanding of the assay will be needed for
animal studies where a variety of tissues will be available vs. human clinical assessment, where typically blood is readily available and other matrices may be limited.
Finally, understanding the purpose of the assay measurement will be critical in
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understanding platforms and sensitivity. For assessment of shortterm increases in
transgene levels, options will be varied. Although if one wants to compare long
term durability of expression, a protein assay for a transgene protein expressed in
liver cell hepatocytes with lifetime of several hundred days may be different than
the requirement to assess transgene protein expression levels in neurons with
potentially a cell lifetime as long as the lifespan of the individual[18].
9.2.6.1 Immunoassays
The most common methodologies for quantitation of proteins are LBA. Formats such
as ELISA. LBAs are precise, accurate and allow, in general, for very specific detection
of a particular protein (Findlay and editor). In addition, the ability to amplify the
signal with ELISA or ImmunoPCR LBA platforms, or detect very low levels of analyte
using luminescence techniques (e.g. ElectroChemical Luminescence) makes immunoassays the most sensitive platform for protein quantitation. Immunoassays are well
suited for the detection and quantification of transgene protein levels in biological
matrices.
However, two key challenges can exist while using immunoassays for gene therapy transgene protein detection. First, while specificity of an immunoassay is
typically high, the ability to distinguish between endogenous levels of a protein
and transgeneexpressed protein if they are very homologous may not be possible.
Of course, no method can distinguish between identical proteins, but in some
cases, the transgene protein sequence may be slightly different than the endogenous protein[2] and the ability to distinguish between them critical for understanding success of the gene therapy treatment.
The second challenge is that immunoassays may be subject to background
interference from the biological matrices. While liquid matrices such as serum,
cerebral spinal fluid, and aqueous or vitreous humor in general to not have significant interference that cannot be resolved[19], tissue homogenates from transgene
protein biodistribution studies may result in sufficient interference to limit the
limit of quantitation of an immunoassay[20].
9.2.6.2 Mass Spectrometry Assays
MS, coupled with liquid chromatography (LC) separation is an ideal platform for
quantifying low molecular weight molecules. And the platform, coupled with protein digestion to peptides, allows this platform to be used to quantify proteins[21].
In general, LC–MS platforms are not as sensitive as immunoassays because of
extensive background in biological matrices. However, use of an immunoaffinity
step to isolate the protein of interest may allow the LC–MS methodology to achieve
similar sensitivities as immunoassays [22]. In general, the specific application
where LC–MS can excel over immunoassays for quantifying transgene proteins
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