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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5665_Библиотеки_им_академика_М_И_Перельмана
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Transgene mRNA Expression Analysis
Venkata Vepachedu and Hsing-Yin Liu
Molecular Biology, Johnson and Johnson Innovative Medicine, Janssen Pharmaceuticals, Spring House, PA, USA
8.1 Purpose of Measuring Transgene mRNA
One important parameter in measuring the efficacy of traditional small‐molecule
medicine is to follow the drug concentration to build a pharmacokinetic (PK) profile. The possible drug metabolites are captured carefully to assess when and
where the functional drug and its metabolites are therapeutic. The pharmacodynamics (PD) studies are employed to correlate the relationship between drug concentration and its biologic effects. GTx is different from small molecule medicine
due to the complexity of the “drug” itself. In an AAV‐mediated GTx, for example,
the direct drug is the DNA packed into the AAV capsid. This packed DNA encodes
the therapeutic payload, including cDNA replacement/gene addition, exon‐
skipping, RNA degradation, or genome editing.[1]. When combined with possible systemic or target‐tissue‐specific deliveries by modifying the AAV capsid,
AAV‐mediated GTx is a very attractive platform for future medicine.
The additional advantages of AAV‐mediated GTx are the rare incident of chromosomal integration and the ability to infect both dividing and nondividing cells.
However, these can also be seen as disadvantages. AAVs enter the host cells and
stay as an episomal form without further replication [2]. This might not be a
problem if the target cells are nondividing cells. If the target cells are rapidly
dividing cells, a number of these episomal AAV DNA will be diluted quickly and
lose its therapeutic efficacy. Hence, monitoring the persistence of AAV genome
and its products is critical for the PK profile.
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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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Despite an elaborate design and engineering process before packing the recombinant DNA into the AAV capsids, there is no guarantee that the intended gene product will be expressed and functional. In addition, the expression levels of the
intended drug product are critical for it to be therapeutic. As mentioned earlier,
AAV‐mediated GTx is flexible to deliver many different drug modalities: (1) gene
replacement or addition (~90%) [3] that produces full‐length or truncated‐
but‐
functional replacement proteins; (2) small interference RNAs (siRNAs) or
microRNAs (miRNAs) to reduce a target messenger RNA (mRNA)[3], and
(3) CRISPR‐Cas9/guide RNA (gRNA) components to perform gene‐editing in the
target cells [4]. Depending on the MOA, the AAV‐mediate GTx can result in
increased or decreased levels of targeted gene product (protein), which often can be
captured at the RNA level as well.
For AAV‐mediated GTx, the PK profile includes the tracking of the drug (AAV
vector genome), the payload (the direct transgene RNA), and final drug product
(transgene product, both the target mRNA species and the translated protein).
The final protein product is the driving force for a successful PD profile[5], and it
will be discussed in separate chapter of this book. Currently, the measurement of
transgene mRNA is performed with quantitative RT‐PCR (RT‐qPCR or RT‐dPCR)
and in situ hybridization (ISH), both will be discussed in this chapter.
8.1.1 Transgene Encodes Therapeutic Protein Entity
The AAV vector has an insert size limitation of ~4.7 kb[2], which translates into
approximately 170
therapeutics, it is critical to quantify the direct transcription product of the AAV
recombinant DNA during the development and preclinical stage. Absolute
RT‐qPCR is commonly used to quantify the expression levels of the target
transgene mRNA. One must keep in mind that the transgene mRNA could be the
direct or indirect product of the AAV‐mediated GTx depending on the modality
type the AAV cassette carries (Figure8.1).
If the replacement protein carried by an AAV‐mediated GTx is a full‐length
endogenous protein, the recombinant DNA sequence in the AAV cassette is likely
significantly different from the endogenous transcript due to codon optimization.
If so, the RT‐qPCR assay development strategy is straightforward to differentiate
the recombinant from the endogenous transcript (Figure8.1a). Depending on the
program, it might be also important to monitor the expression levels of both
endogenous mRNA (if any) and transgene mRNA.
Not all AAV‐mediated protein replacement therapies are designed to produce
full‐length proteins. To treat diseases like Duchenne muscular dystrophy (DMD),
a truncated protein produced by antisense oligo‐directed exon‐skipping mRNA is
sufficient to restore the dystrophin (DMD gene product) function. In this case, it
kDa of protein size. Regardless of the MOA of the specific AAV
8.1 Purpose of Measuring Transgene mRNA 195
A.
Direct transcript Final transcript
B.
C.
D.
E.
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Figure8.1 The AAV-mediated GTx may deliver direct transcript from the AAV vector and
then the final transcripts mediated by the direct transcript. See text for detail.
is critical to monitor both exon‐skipped transgene mRNA and antisense oligos
(which will be discussed in Section8.1.2)[6] to further demonstrate the MOA as
well as the accuracy of the exon‐skipping product (Figure8.1c).
Alternatively, an AAV carrying artificial miRNA targeting mutant Huntingtin
(mHTT) mRNA results in decreased levels of mHTT mRNA, but not wild‐type
HTT (wtHTT) is currently on clinical trial for treating Huntington disease (HD).
The key pathology of HD is caused by the aggregation of mHTT protein in the
brain. This specific AAV5‐miHTT binds to the mutant exon1 of the mRNA to
inhibit it from being translated into the toxic mHTT (Figure 8.1d). Because
wtHTT is an essential protein, it is important to monitor the expression levels of
both wild‐type and mutant HTT mRNA during the development stage[7]. For
AAV platform that carries antisense oligonucleotides to mediate the gene silencing, carefully monitoring the reduction of target mRNA is obviously the key
(Figure8.1b). Lastly, dual vector systems to deliver CRISPR‐Cas9/gRNA gene
editing therapy are on the rise[8]. Depending on the MOA of specific GTx, quantifications of RNA species derived from gene editing components (the Cas9nuclease mRNA and gRNA), as well as the homologous recombination template DNA,
may be required (Figure8.1e).
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8.1.2 Transgene Encodes Other Entities
As mentioned previously, in addition to delivering mRNA that generates gene
replacement protein, the AAV‐mediated GTx can also deliver oligonucleotides
that produce specific‐splice variant mRNA, or gene editing machinery, including
the nuclease and the gRNA.
While it is critical to track the final gene product quantity of the specific‐splice
variant mRNA or edited mRNA, the expression levels of these oligonucleotides
and the mRNA encoding the editing nuclease are also important per Food and
Drug Administration (FDA) guidance[9]. For unconventional GTx like these, it is
essential to monitor all AAV‐generated products: the direct RNA species (mRNA,
siRNA, miRNA gRNA, etc.), and the final RNA product (spliced mRNA, exon‐
skipped mRNA, edited mRNA, etc.), during the development, preclinical,
nonclinical, and clinical stages, to ensure intended and efficacious gene products
are generated in the target tissue, as well as monitoring the off‐target toxicity
associated with these RNA species. The techniques to quantify oligonucleotides
will be covered in Section8.2.1, and the detection and quantification of gene editing at the genome level will be discussed in separate chapter.
8.2 Technologies toQuantify Transgene Expression
inTissues
8.2.1 RT-qPCR or RT-dPCR
The discovery of critical reagent thermostable DNA polymerase gave a breakthrough
in the PCR from a strenuous frequent addition of Klenow fragment to a continuous
reaction and made it possible for universal application[10]. Development of programmable thermostats transformed the process of PCR from a three water bath to a
single dry bath reaction[11]. Subsequently, real‐time PCR machines hit the market
in 1996 [11] allowing the detection of amplification in real time up to
48 samples in one run for detection and quantification of gene or sequence of interest, i.e. quantitative PCR or qPCR. By reverse transcription as the first step to make
cDNA and then by qPCR of cDNA, i.e. RT‐qPCR, levels of RNA or transgene expression are quantified. The terminology used for qPCR and RT‐qPCR has been discussed
briefly in a separate chapter. As per Minimum Information for Quantitative PCR
Experiments (MIQE) [12] guidelines, we used the terms qPCR for DNA and
RT‐qPCR for mRNA quantitation by real time PCR. RT‐qPCR is the gold‐standard
technique for either absolute or relative quantification of transgene expression levels
in mammalian or human tissues (or body fluids such as saliva, tears, or serum). Thus,
except for the conversion of RNA to cDNA by using a reverse transcriptase (RT step),
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the technology of qPCR and RT‐qPCR remained the same. The development of
single‐step RT‐qPCR and efficient reverse transcriptases have made the RT‐qPCR
process more easy and simpler [13]. Digital PCR (dPCR) is also a quantitative PCR
method but a novel advancement over qPCR termed by some as next‐
qPCR technology that involves absolute quantitation of nucleic acid target sequences
without a standard curve. In brief, the main difference between real-time based
qPCR and dPCR is that in qPCR the amplification reaction is monitored throughout
the process and quantification is based on the fluorescent signal analysis at the exponential phase, whereas in dPCR the sample is distributed in partitions and signals are
measured at the end to quantify based on the number of positive partitions over the
total[14]. The details and a comparative discussion will be performed while discussing the quantification strategies. Factors affecting the accurate quantification of
transgene expression include the quality of RNA, assay design, and analysis. Hence,
the technologies/methods available (Figure8.2) and in use for
1) Extraction/purification
2) Quantification and quality check
3) Reverse‐transcription and qPCR assay
4) Analysis of the raw data
will be discussed.
8.2.1.1 RNA Extraction (Separate vs. DNA/RNA Co-extraction), Quality
Testing, and Quantification
Total RNA extraction of large number of samples has become easier to perform with
the development of a single‐step extraction method using guanidium
phenol–chloroform extraction technique under acidic conditions[15]. This also led
to the production of several kits, including column‐based kits globally. Astudy was
performed on the efficiency of six RNA extraction methods using commercially
available kits, RNAqueous kit, micro‐to‐midi total RNA purification system,
NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and
TRIzol LS reagent, were evaluated on blood and seven tissues showed acceptable
performance of all methods for RT‐qPCR [16]. A Trizol‐based method showed
slightly better performance in this study. However, successful extraction and purification of either DNA or RNA depends on the quality of homogenization of the
tissue for the release of RNA and removal of contaminating DNA. Currently, high‐
quality RNA extraction kits are available from various vendors, such as ThermoFisher
scientific and Qiagen, for blood and other tissues or prokaryotes. However, gene/cell
therapy projects (AAV vectors or LNP) involving PK/PD studies require the
extraction of RNA from large number of samples that require high‐throughput platforms and kits for the same. New kits and technologies or platforms have been
generation
thiocyanate–
Total RNA
emulsion and generate droplets,
to make 20,000 droplets/sample.
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extraction
Quality check-
integrity (RIN)
purity and
RNA
quantification
2
run
RNA samples
Microfluidic array
based partitioned plate
based digital PCR
Mix the MM and RNA,
QIAcuity Eight
load the wells.
The sample
spreads into the
microwells
3
Mix MM and RNA and oil
Droplets are transferred to a
96-well plate for PCR in a
thermal cycler
Following RT-PCR, plates are
placed in droplet reader for
counting +Ve and –Ve droplets
in each sample.
Add MM & RNA in
96/384 well plate or
strip tubes
1
qPCR based
Place on
instrument and
start program
Single step RT-
qPCR master mix,
primer/probes
A. Nanoplate with 96 well; B. single well
detail; C. cross section view of the
partitions (from Qiagen)
Place on a
digital PCR
instrument and
4
Data analysis and reporting
Figure8.2 Schematic of RT-qPCR/RT-dPCR for gene expression analysis in GTx pharmacokinetic studies. All cartoon pictures are
drawn on PowerPoint.
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introduced for high‐throughput extraction of DNA/RNA with greater simplicity, cost
saving, and maintaining quality and yield. The filter plate and nucleic acid extraction
spin column offer for high‐ and low‐throughput nucleic acid purification[17]. The
introduction of magnetic bead‐based procedure improved the high‐throughput
extractions as filter plate or spin‐column based protocols may lead to clogging when
used for tissues. New platforms along with kits to use have been introduced for
semi‐automated extraction platforms for 24, 48, or 96 samples in a single run. Some
of the popular automated instruments are Roche Magnapure, Qiagen‐Qiacube,
ThermoFisher– Kingfisher and Promega‐Maxwell.
However, they supply separate kits for DNA or RNA for different tissues/blood.
The quality and yield vary between different kits for each sample type. However,
the yield of the automated extraction kits also depends on the extent of sample
(tissue) homogenization either by manual or high‐throughput instruments such
as a Tissue lyser or Omniruptor. The selection of proper lysis protocol and homo
genizer is essential. DNase treatment is very crucial in RNA purification and some
automated instruments/kits tend to show slightly higher DNA contamination
from specific tissues[18]. For most of the gene/cell therapy projects the available
automated instruments and kits are useful in the extraction of quality RNA with
high RIN (RNA Integrity Number), a user‐independent algorithm for the assignment of integrity numbers to an RNA sample using a scale of 1–10.
8.2.1.2 Co-extraction ofDNA and RNA fromsame Sample
Blood/tissue collection limitations from mice[19, 20], low concentration of GTx
drug target in clinical samples (shedding) and for better comparable results[21]
some protocols combining the DNA and RNA kits have been designed and
tested[22]. In this, the lysis buffer which is usually discarded after binding DNA
to resin was processed for RNA extraction. Also, there are kits developed for the
co‐extraction of DNA and RNA from the same sample using magnetic bead separation and precipitation at different stages. Comparative assessment of those kits
was performed from environmental samples and biological tissues [23]. It was
found that though the yields vary, the quality of the RNA is acceptable for sequencing or RT‐qPCR. Simultaneous extraction of DNA and RNA from Ambion’s
ToTally RNA kit from tumor, spleen, and other tissues seems to show quality
results and is also compatible with Kingfisher automated systems[24]. However,
co‐extraction of DNA/RNA is not yet widely used in gene/cell therapy studies for
PK/PD assays probably due to higher quantities needed for validated quantitative/
relative quantitative assays per non‐GLP and GLP requirements. As the GxP studies for biodistribution require processing of large number of samples, automation‐
friendly kits for co‐extraction will encourage wide usage.
-
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8.2.1.3 Quantification and Quality Testing oftotal RNA
inPurified Extracts
For academic or diagnostic project purposes, purified DNA or RNA can directly go
for target detection or quantification by qPCR/RT‐qPCR [25]. However, AAV‐
based or other GTx/CTx studies have stringent validation requirements for PK
analysis. To meet this, the samples need to have equal total RNA concentration to
avoid any matrix effect. Hence, the guidelines for PK analysis in AAV or other GTx
studies suggest quantitating the total RNA extractions from samples and normalize them before performing drug or target RNA quantitation by RT‐qPCR[26]. For
total RNA quantification, the instruments Nanodrop/Denovix/UV–VIS spectrophotometer[26], Qubit, Agilent TapeStation, and Promega Glomax, etc. are popularly used in different labs globally (Table 8.1). However, specificity of Nanodrop
or Denovix is very less, compared to the other three instruments which are fluorescent based (Qubit & Glomax or electrophoresis based– TapeStation). Because,
both DNA/RNA, nucleotides, and several aromatic compounds also absorb at
260/280
nm. In addition, Agilent TapeStation and Promega Glomax offer high‐
throughput technologies more suitable for the analysis of large number of samples. Nanodrop is also used to test the quality based on A260/A280 nm absorbance
as explained in the next section.
The easiest and routine way to confirm the purity is by taking absorbance at
260/280
nm using a spectrophotometer or Nanodrop[27]. As RNA is relatively
labile and prone to degradation, additional care is taken to preserve and confirm
its integrity. This includes optimizing extraction conditions and confirmation of
RNA quality in the samples– random or individual as preferred. Because mRNA
is expressed in low quantities, RNA integrity in eukaryotic cells is commonly
determined by quantifying the more abundant 18S and 28S ribosomal RNA
(rRNA) transcripts[28]. The quality of RNA is proportional to the percentage of
full‐length transcripts. In the earlier days, the quality used to be assessed by agarose or capillary electrophoresis [29]. However, this is a bit subjective. Using
Table8.1 Commonly used instruments fortotal RNA quality check and quantification
inGTx studies.
Type Model Manufacturer
Spectrophotometer DeNovix Denovix
Fluorescent‐based Qubit Promega
Electrophoresis‐based Bioanalyzer (chip‐based) Agilent
NanoDrop ThermoFisher
Glomax (high throughput) Promega
TapeStation (high throughput) Agilent
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microcapillary electrophoresis, Agilent technologies have developed a user‐
independent, automated procedure for standardization of RNA quality control
that allows the calculation of an RIN[30]. This is widely applied in research and
clinical studies to confirm the quality of RNA before going for RT‐qPCR. The
Agilent 5200 Fragment Analyzer system and Agilent ProSize data analysis software use an RNA quality number (RQN) as a quality metric indicator. ProSize
considers the entire electropherogram to give a RQN number [31]. However, in
another technical note they published in the recent Agilent platforms total electropherogram is taken into consideration to provide RIN or RQN and both values
are interchangeable [32]. A method to measure spatial RNA integrity number
(sRIN) in situ from a single tissue section suitable for clinical samples has been
developed as a new tool[33].
8.2.1.4 Quantification Using DNA vs. RNA Standards
Quantification of target RNA by RT‐qPCR requires a standard curve and needs
preparation of standards. However, the RT‐dPCR is gaining more preference due
to some advantages especially as this does not need a standard curve. The term
“digital PCR” was coined by Vogelstein and Kinzler in 1999[34]. However, in the
case of digital PCRs, they are of several varieties depending on the manufacturer.
Bio‐Rad’s digital droplet PCR became very popular. The general parameters for
real‐time RT‐qPCR guidelines were provided in MIQE 2009[12]. For digital qPCR
also MIQE guidelines were published initially in 2013 and an update was provided in 2020[35, 36]. Though RT‐dPCR has several advantages over RT‐qPCR as
listed in Table8.2, there is a need to test the reliability of the absolute copy number output and differentiation of positive and negative reaction units.
Some of the commercially available digital PCR systems are listed in Table8.3.
8.2.1.5 Assay Qualification/Validation and Report
Due to the lack of specific regulations from FDA, European Medicines Agency
(EMA), or other regulatory agencies, the guidelines from different forums, white
papers, and MIQE [12, 33] are followed for assay qualification/validation and
reporting (see Chapter10. Some of the points were discussed in separate chapter
and will be briefly touched on here. Assay qualification of RT‐qPCR or RT‐dPCR
assays for clinical trials is required to meet same acceptance criteria for assay qualification and validation in general except that while performing the assay, RT‐dPCR
does not require a standard curve. In either way, the RNA extraction method, and its
stability are equally important. The general criteria recommended and followed for
validation and acceptance criteria for RT‐qPCR and RT‐dPCR are listed in Table8.4.
How extensive a method needs to be validated depends on how much uncertainty exists in the study samples and regulatory needs. Though a standard curve
is not needed, RT‐dPCR also requires calibrators/quality controls with the range
expected in the study samples[37].
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Table8.2 Comparative overview ofRT-qPCR vs. RT-dPCR.
Item Real Time Digital
Principle Quantification in real time as it
occurs after every cycle. Data
collected during exponential (log)
phase– PCR product formed is
equal to the amount of template.
Partitioning of the PCR reaction
into thousands of individual
reaction vessels prior to
amplification. Microfluidics
technology is used for
digitization. Endpoint data
acquisition.
Quantification Relative quantification with a
reference gene or absolute
Absolute quantification possible
without a standard curve.
quantification with a set of
standards.
Quantification Ct Value is the PCR cycle
number at which a specific
sample reaches fluorescent
intensity above the set threshold
background. A standard curve is
constructed with the Ct values of
a set of standards of known copy
numbers and used in downstream
quantitation or detection in
After PCR, the target copy
number is calculated based on
the fraction of positive/negative
microreaction wells/droplets.
APoisson distribution co‐efficient
is used to correct the copies per
droplet using the equation=
−ln(1
− p), where p= fraction of
positive droplets
samples. No post‐PCR processing
required.
Precision Good at higher copy number in
the range.
Higher precision can be obtained
with a greater number of
replicates and is better than
real‐time RT‐qPCR.
PCR inhibitors No option to specially reduce
PCR inhibitor other than
Tolerance to PCR inhibitors is
more due to digitalization.
including a positive control.
Cost Platforms are already installed in
the labs worldwide and less
Initial startup slightly more
expensive.
expenditure.
Upper limit of
quantitation
(ULOQ)
Lower limit of
quantitation
Upper limit of quantification
8
is10
copies in general.
Varies (3 and above copies/
reaction) depending on the target.
Upper limit of quantitation with
current platforms is 105 copies as
of 2022.
Reproducibility is higher at
LLOQ.
(LLOQ)
Multiplexing Higher capability Limited
Samples/plate 96 or 384well format 96well format maximum
Run time Can go to <90 min 2 h or higher
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