Regulatory Guildilens For Preclinical Tools To Study Biodistibution of RNA Therapeutucs

Advanced Drug Delivery Reviews 184 (2022) 114236
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/adr
Regulatory guidelines and preclinical tools to study the biodistribution

of RNA therapeutics
P. Vervaeke a, S.E. Borgos b, N.N. Sanders c,1, F. Combes d,⇑,1
a
Laboratory of Gene Therapy, Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium
b
SINTEF Industry, Dept. of Biotechnology and Nanomedicine, Research Group Mass Spectrometry, Sem Sælands v. 2A, N-7034 Trondheim, Norway
c
Laboratory of Gene Therapy, Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium
d
SINTEF Industry, Dept. of Biotechnology and Nanomedicine, Research Group Mass Spectrometry, Sem Sælands v. 2A, N-7034 Trondheim, Norway
a r t i c l e i n f o a b s t r a c t
Article history: The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the
Received 20 December 2021 beginning of a new chapter in modern medicine. However, the rapid rise of mRNA therapeutics has
Revised 9 February 2022 resulted in a regulatory framework that is somewhat lagging. The current guidelines either do not apply,
Accepted 23 March 2022
do not mention RNA therapeutics, or do not have widely accepted definitions. This review describes the
Available online 26 March 2022
guidelines for preclinical biodistribution studies of mRNA/siRNA therapeutics and highlights the relevant
differences for mRNA vaccines. We also discuss the role of in vivo RNA imaging techniques and other
Keywords:
assays to fulfill and/or complement the regulatory requirements. Specifically, quantitative whole-body
Guidelines
Biodistribution
autoradiography, microautoradiography, mass spectrometry-based assays, hybridization techniques
Distribution (FISH, bDNA), PCR-based methods, in vivo fluorescence imaging, and in vivo bioluminescence imaging,
RNA therapeutic are discussed. We conclude that this new and rapidly evolving class of medicines demands a multi-
mRNA vaccine layered approach to fully understand its biodistribution and in vivo characteristics.
mRNA Ó 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
siRNA creativecommons.org/licenses/by/4.0/).
Vaccine
Lipid nanoparticle
LNP
Imaging
FLI
BLI
QWBA
RT-qPCR
FISH
bDNA
Mass spectrometry
MS
Regulatory
Onpattro
Patisiran
Givlaari
Givosiran
Leqvio
Inclisiran
Oxlumo
Lumasiran
Comirnatmy
Pfizer/BioNTech
Spikevax
Moderna
⇑ Corresponding author.
E-mail addresses: [email protected] (N.N. Sanders), [email protected] (F. Combes).
1
These authors contributed equally.
https://doi.org/10.1016/j.addr.2022.114236
0169-409X/Ó 2022 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P. Vervaeke, S.E. Borgos, N.N. Sanders et al. Advanced Drug Delivery Reviews 184 (2022) 114236
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Regulatory framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Regulatory guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Validation of analytical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Choice of animal species and animal model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.3. Number of animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.4. Duration of longitudinal animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.5. Minimal tissue panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.6. Compliance to good laboratory practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Techniques used for authorized RNA therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Quantitative whole-body autoradiography and microautoradiography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Mass spectrometry-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Hybridization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Other techniques that could reach EMA/FDA requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. Reverse transcription quantitative polymerase chain reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Fluorescence and bioluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.1. In vivo bioluminescence imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.2. In vivo fluorescence imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1. Introduction Consequently, this FDA guidance document contains the most

up-to-date guidelines on mRNA vaccines and will be further
The success of the messenger RNA (mRNA)-based COVID-19 updated after the pandemic [16]. Additionally, new mRNA-
vaccines of Moderna and Pfizer/BioNTech marks the beginning of specific guidelines are expected by the WHO [17] and possibly also
a new chapter in modern medicine. Within weeks, any therapeutic national regulatory agencies, such as EMA and FDA [18]. All biodis-
protein of choice can now be encoded on mRNA, encapsulated in tribution guidelines (summarized in Table 3) are nonbinding and
lipid nanoparticles (LNPs) and be supported by preclinical data. generally less strict than those for e.g., toxicology studies, but
Within months, the mRNA COVID-19 vaccines were tested in clin- applicants should consider that additional, binding national and
ical trials and within a year, were brought to the market [1–3]. The international legislation may apply.
COVID-19 pandemic is considered a public health emergency by
the European Medicines Agency (EMA) and the U.S. Food and Drug
Administration (FDA), because the immediate availability of vacci- 2. Regulatory framework
nes outweighs the risk associated with less comprehensive phar-
maceutical and clinical data at the moment of authorization [3– The goal of preclinical biodistribution studies is to characterize
5]. Consequently, SARS-CoV-2 vaccines became eligible for condi- the presence, persistence, and clearance of the drug at the molecu-
tional marketing authorization in Europe and emergency use lar level both in target tissues and an array of non-target tissues
authorization in the United States (US) [3–5]. The shortened time- [7,9,10,14]. They are an important component of preclinical phar-
line associated with these approvals contrasts strongly with the macokinetic studies and help interpret nonclinical pharmacology
mean duration of 10 years to develop and authorize a new drug and toxicology findings [8,9,11]. The requirement for preclinical
[6]. Moreover, the rapid rise of mRNA therapeutics has resulted biodistribution studies to initiate first-in-human studies is decided
in a regulatory framework that is somewhat lagging. The current by regulatory agencies on a per-product basis. However, the default
guidelines either do not apply, do not mention RNA therapeutics, approach differs for RNA therapeutics and mRNA vaccines (Table 1).
or do not have widely accepted definitions [7–11]. For RNA therapeutics, EMA advises that biodistribution studies are
In this review, ‘‘RNA therapeutics” refers to small interfering always performed, unless the design and type of the RNA therapeu-
RNA (siRNA) and mRNA. It is important to note that prophylactic tic justifies otherwise [7,8,12]. FDA also advises that biodistribution
and therapeutic vaccines against infectious diseases are currently studies are performed, but only for new vector classes and when
not considered ‘‘gene therapy medicinal products” or ‘‘gene ther- significant changes are made in vector backbones, formulations,
apy products”, according to EMA and FDA, respectively routes of administration, dose levels, and dosing schedules [9]. In
[8,9,12,13]. However, the guidelines for vaccines and RNA thera- contrast, mRNA vaccines do not require pharmacokinetic studies
peutics are similar for many of the discussed points. This review (which encompass biodistribution studies) to initiate first-in-
describes the guidelines for biodistribution studies of RNA thera- human studies (both in Europe and the US) unless the vaccine uses
peutics and highlights the relevant differences for mRNA vaccines. novel adjuvants, formulations, additives, or routes of administra-
We also discuss the role of in vivo RNA imaging techniques to fulfill tion [11,15]. Because of the per-product-approach, developers of
and/or complement the regulatory requirements issued by FDA RNA therapeutics or mRNA vaccines are strongly advised to request
[9,10], EMA [7,8], the International Council for Harmonisation assistance from the relevant regulatory body before initiating pre-
(ICH) [11], and the International Pharmaceutical Regulators clinical studies [7–10,19]. Moreover, the specific requirements for
Programme (IPRP) [14]. For vaccines, EMA refers to the WHO how the biodistribution study should be designed will be tailored
guidelines [15], whereas FDA has issued its own guidance to each product. For example, RNA therapeutics are often delivered
document in response to the COVID-19 pandemic [16]. through some carrier or delivery system, such as lipid nanoparticles
2
Table 1
Different requirement for biodistribution studies for RNA therapeutics and mRNA vaccines.
RNA therapeutics
Europe
European Directive 2001/83/EC [12], ‘‘Biodistribution studies shall include investigations on persistence, clearance and mobilisations. Biodistribution studies shall
additionally address the risk of germline transmission.”
United States
FDA (2013) [9], ‘‘Prior to administration in humans, biodistribution studies should be considered for:
a. Investigational gene therapy products that belong to a new vector class.
b. Established vectors with significant changes in the vector backbone.
c. Established vectors with a significant formulation change.
d. Established vectors with a significant change in route of administration.
e. Established vectors with a significant change in the dosing schedule and/or the vector dose levels.”
mRNA vaccines
Europe and United States

WHO (2005) [15], ‘‘Pharmacokinetic studies . . . are normally not needed. Distribution studies should be considered in the case of new formulations, novel adjuvants
or when alternative routes of administration are intended to be used. . .”
(LNP) or polymers. Consequently, preclinical biodistribution stud- Both for RNA therapeutics and mRNA vaccines, the preclinical
ies may be requested for the therapeutic RNA construct, the car- product should reflect the intended clinical product and its applica-
rier/delivery system, individual components of the carrier/ tions as much as possible. However, deviations can be justified. For
delivery system, and the combined final product [8,11,14]. For example, adjusting dosages to different species or using a different
mRNA-based therapeutics, biodistribution of the produced protein RNA molecule in a certain carrier [7–11,14–16]. Because changing
product should also be investigated. the coding sequence of the mRNA is unlikely to have a significant
Biodistribution studies can in some cases be avoided. For exam- impact on the physicochemical properties and distribution of
ple, in the preclinical studies of the Pfizer/BioNTech COVID-19 vac- mRNA-LNPs, luciferase-encoding mRNA may be used to monitor
cine [20] and the Moderna COVID-19 vaccine [21], no the distribution of a novel formulation. This justification was pro-
biodistribution studies were included for the final mRNA-LNP for- vided for the COVID-19 vaccines of Pfizer/BioNTech and Moderna
mulation. Instead, these applications relied on biodistribution [20,21]. Finally, although the marketing authorization of these
studies from LNP formulations encapsulating a different mRNA vaccines was conditional, no additional non-clinical in vivo
nucleoside-modified RNA (modRNA) sequence [20,21]. Similarly, data (including biodistribution) was requested in the European Pub-
the guidelines also imply that no additional biodistribution and lic Assessment Reports, indicating that the provided data was suffi-
pharmacokinetic studies are required for new mRNA vaccines cient for a future full marketing authorization [4,20,21].
which only alter the modRNA component, but use the same carrier
and route of administration as either the Pfizer/BioNTech or the 3. Regulatory guidelines
Moderna COVID-19 vaccine [15]. In theory, applicants from other
companies can thus avoid biodistribution studies by referring to 3.1. Validation of analytical techniques
the data in the Pfizer/BioNTech and Moderna applications. In prac-
tice however, access to the experimental data may be subject to The analytical techniques that are used in preclinical studies of
company secrecy. In addition, FDA guidelines even state that new RNA therapeutics should be validated [8,10,11,14]. This also applies
vaccines using the COVID-19 vaccine (mRNA) platform technolo- to mRNA vaccines [15,16]. Applicants must demonstrate that the
gies can rely on the existing toxicology data [16]. The same ratio- used techniques, procedures, equipment, and materials are appro-
nale applies to non-vaccine mRNA therapeutics, i.e., priate to detect the target (i.e., the RNA product, a carrier compo-
biodistribution studies can be avoided when only the protein cod- nent, or the expressed protein) at the molecular level and in the
ing sequence of mRNA is changed and adequate justification for relevant biological matrix [8,9,15]. This can be demonstrated either
not performing the study is provided [8,10,11]. through own experimental data or by referring to published data.
Apart from the RNA component, novel carrier components such Guidelines on validation of analytical techniques are available from
as the lipids in LNPs need to be supported by individual preclinical multiple agencies [24–27]. Limits of quantification should be speci-
studies as well [7–10]. For example, the LNP formulation used in fied, as well as techniques used to obtain test samples and the order
the Pfizer/BioNTech COVID-19 vaccine contains four lipids, of in which samples are obtained. The latter is important to prevent, for
which only the ionizable lipid (ALC-0315) and the polyethylene example, cross-contamination between samples [8,10,11,14].
glycol (PEG)-phospholipid conjugate (ALC-0159) were novel com- Regarding DNA detection, guidelines from EMA, ICH, IPRP, and
ponents. Indeed, cholesterol and distearoyl phosphatidylcholine FDA refer to quantitative Polymerase Chain Reaction (qPCR) [7,9–
(DSPC) comply with the European Pharmacopoeia and are used 11,14]. Note that the European guidelines use the term ‘Nucleic
in several already approved products [20]. More specifically, the Acid Amplification Testing’, which is a broader term and might
provided justification referred to the use of DSPC in the LNP of include alternative methods such as loop-mediated isothermal
OnpattroÒ, which is an EU-approved siRNA drug administered amplification [8]. EMA, ICH and IPRP guidelines add Reverse Tran-
intravenously and at a much higher dose than the intended intra- scription qPCR (RT-qPCR) for RNA [8,11,14]. ICH also lists tech-
muscular dose of the Pfizer/BioNTech COVID-19 vaccine [22]. In niques that can be used to monitor the RNA product or the
addition, the structurally related dioleoyl phosphatidylcholine expressed protein product in nonclinical biodistribution studies:
(DOPC) is also used in intramuscular products and approved by enzyme-linked immunosorbent assay (ELISA), immunohistochem-
the EU [20,23]. EMA therefore only requested new studies for the istry (IHC), western blot, in situ hybridization (ISH), digital PCR,
novel components (ALC-0315 and ALC-0159) and their combined flow cytometry, in vitro and in vivo imaging techniques and ‘‘other
use in fully formed mRNA-LNPs [20]. evolving technologies” (which still require validation) [11].
3
3.2. Choice of animal species and animal model icology, which advise to use 10 rodents per sex and per experimen-
tal group [15]. To reduce the number of animals, endpoint analysis
The chosen animal species used in preclinical studies on RNA can sometimes be complemented or replaced by non-invasive
therapeutics should be as biologically relevant as possible and imaging methods in the same animal. This is important for mRNA
show a pharmacological response that is similar to the expected therapeutics, which typically require many assessments at short
response in humans. This also applies to mRNA vaccines [7– interval timepoints post-injection. The number of animals can also
11,14–16]. For siRNA therapeutics, this means that the animal spe- be reduced by performing multiple studies within one animal. To
cies should ideally express an mRNA target that has an identical allow correlation of possible toxicity to the presence or absence
sequence to that expressed in humans. For mRNA therapeutics, this of the investigated compound, it is recommended to use the same
suggests that the delivered mRNA should be translated in a similar animal model in both toxicology and biodistribution studies
manner (i.e., amount, duration, and distribution) to that expected [8,9,14]. Thus, combining aspects of both studies in one experi-
in humans, and that the animals should show a similar biological ment avoids unnecessary use of animals and provides a better cor-
response to the produced protein product. To achieve comparable relation between toxicology and biodistribution [8,11]. In general,
translation kinetics of mRNA, the innate immune response is also both genders should equally be represented in the experiments,
an important factor to consider, because a strong response can sig- but deviations are allowed when adequately justified [8,10,11,14].
nificantly reduce mRNA expression [28–31]. This might imply that
rodents are not suited for all RNA therapeutics. For example, mice
have a toll-like receptor 8 (TLR8) that functions differently from 3.4. Duration of longitudinal animal studies
the TLR8 in humans [32]. This receptor plays an important role
in triggering an innate immune response, upon recognition of for- The duration of longitudinal animal experiments depends on
eign single-stranded uridine-rich, unmodified RNA [28]. This the RNA therapeutic, the dose, the encoded protein, the carrier,
response can potentially lead to differences in translation kinetics, and the route of administration [7–10]. For example, the biolumi-
and toxicological effects, resulting in unexpected drug responses in nescence of intravenously administered LNP-mRNA (encoding luci-
humans [29–31,33]. Additionally, recent reports demonstrate that ferase) was no longer detectable in the liver after 3 days but
mice, and even non-human primates, do not reliably predict remained detectable at the injection site for up to 7 days [37].
human systemic inflammatory events after RNA administration. When the same product was injected subcutaneously or intrader-
Compared to humans, it was shown that the expression levels of mally, no signal was seen in the liver but bioluminescence at the
anti-inflammatory interleukin-1 receptor antagonist (IL-1ra) were injection sites endured for 6 and 10 days, respectively [37]. In con-
much higher in mice, both at baseline and after multiple RNA doses trast, intradermal administration of self-amplifying RNA results in
[34]. Similarly, non-human primates had a higher IL-1ra response observable bioluminescence for up to 28 days post-injection [29].
at all RNA dose levels (but not at baseline), while the pro- To address these marked differences between RNA platforms,
inflammatory IL-1b was lower than in humans. These data support EMA and IPRP specify that the study should continue until the sig-
the use of adapted models, such as IL-1ra knock-out mice for the nal becomes undetectable or until a long-term plateau phase is
evaluation of RNA therapeutics and vaccines [34]. reached [8,14]. For modRNA or unmodified mRNA, an appropriate
The used animals can be wild-type, immunocompromised, duration ranges from a few days to a (few) week(s), depending on
knock-out, knock-in, humanized or transgenic animals [8]. In fact, the formulation and route of administration. For self-amplifying or
the use of disease/injury models is encouraged to obtain a better trans-amplifying mRNA, this period is more likely to approach sev-
estimation of the risk–benefit ratio of testing the RNA therapeutic eral weeks. Similarly, subcutaneously injected siRNA can have a
in humans and to improve the biological relevance of the preclin- long half-life, with persistence of up to 40 days being reported
ical data [8,9]. An example of a deviation from wild-type rodents [38]. Newer RNA platforms, such as circular RNA have demon-
are the preclinical studies for COVID-19 vaccines. SARS-CoV-2 uses strated in vitro and in vivo expression profiles of up to a week
human Angiotensin Converting Enzyme 2 (hACE2) to infect cells. [39,40]. Not only the type of therapeutic RNA will influence the
Since rodents do not express hACE2, transgenic hACE2-positive duration of the study, but also the encoded protein product. For
mice and non-human primates were used [20]. Another approach example, proteins with short half-lives are expected to disappear
is to use a mouse adapted SARS-CoV-2 virus [21], or to select alter- shortly after the mRNA translation stops. On the other hand, stable
native SARS-CoV-2 infection models, such as cats, ferrets, and ham- proteins can persist for a longer period, which should be consid-
sters [35,36]. ered when designing a preclinical study. When mRNA is used to
When a single animal species cannot answer all preclinical deliver CRISPR/Cas9 or other gene-editors, preclinical studies will
questions, a panel of studies in various species should be used have to be conducted much longer. Additionally, the risk of vertical
[8,11]. Furthermore, regulatory agencies also consider practical germline transmission of the induced genome modification must
limitations. For example, the small size and short lifespan of be examined. More details on guidance for products with
rodents can be an issue. Similarly, studies in non-human primates genome-altering effects or expected long-term persistence, can
can have sample size limitations and require qualified facilities, be found in FDA and EMA guidelines [8,10]. For vaccines, no appro-
personnel, and expertise [8]. Importantly, preliminary biodistribu- priate duration is mentioned [15,16]. Finally, the elimination pro-
tion studies can help identify relevant species for subsequent phar- files of carrier components, such as ionizable lipids in LNPs
macokinetic, pharmacodynamic, and toxicology studies, by should be considered. Especially with repeated dosing (e.g., long-
determining intracellular gene delivery efficiency or assay method- term protein replacement, (booster) vaccination), accumulation
ologies [11]. can occur. It has been demonstrated that cationic lipids, which
can cause inflammatory and hepatic toxicity, adsorb serum pro-
3.3. Number of animals teins, aggregate and accumulate significantly in the lung, liver
and spleen [41,42]. This is less pronounced (but not completely
FDA and ICH advise to use at least 5 animals per sex per group abolished) with next-generation ionizable lipids [43]. Additionally,
per sacrifice timepoint for rodents and correspondingly 3–5 ani- the use of biodegradable lipids can aid in reducing accumulation of
mals for non-rodents [10,11]. EMA does not specify numbers in these drug components [44,45]. Still, these effects should be con-
their guidelines [7,8], nor does WHO in the vaccine guidelines sidered when determining an appropriate duration of a longitudi-
[15]. Remarkably, this is in contrast with WHO guidelines for tox- nal study.
4
Table 2 Table 3
Minimal tissue panels to be examined in preclinical biodistribution studies, according Guidelines for preclinical biodistribution studies of RNA therapeutics and mRNA
to FDA, ICH, IPRP (identical to FDA’s panel) and EMA.* vaccines.
FDA/IPRP ICH EMA* 1. Applicants are strongly advised to request assistance from the relevant
regulatory body before initiating preclinical studies.
Blood Blood Blood smears
2. Biodistribution studies for RNA therapeutics should encompass both the
Brain Brain Brain (coronal sections at three levels to
RNA molecule(s), the individual components of the carrier, the
include cerebrum, cerebellum and brain
combined RNA-carrier drug, and the produced protein.
stem)
3. In all preclinical studies, the administered drug should reflect the
Gonads Gonads Epididymides, Ovaries, Seminal vesicles
intended clinical product as much as possible, including quality aspects,
(rodents), Testes
dose, dosing regimen, formulation, and route of administration.
Heart Heart Heart
Deviations are allowed when adequate justification is provided.
Injection Injection site(s) Application site (when relevant)
4. Applicants can avoid biodistribution studies by referring to previously
site(s)
performed studies for identical components, dosing, routes of
Kidneys Kidney Kidneys and ureters
administration, etc.
Liver Liver Liver
5. Analytical techniques must be validated. This can be done by referring
Lung Lung Lungs with bronchi and bronchioles
to previous validation or by providing experimental data. Dedicated
Spleen Spleen Spleen
guidelines are available.
Adrenal gland Adrenal glands
6. The experimental animal species and disease models must be as
Spinal cord Spinal cord
relevant as possible to the expected situation in humans. When
(cervical, thoracic,
necessary, multiple species/models can be used.
lumbar)
7. FDA and ICH advise to use at least 5 animals per sex, per experimental
Aorta
group, and per sacrifice timepoint for rodents. For non-rodents, 3–5
Eyes and optic nerves
animals are advised. Biodistribution studies must include both genders,
Gallbladder (when relevant)
but deviations are possible when adequately justified.
Gross lesions
8. The appropriate duration will depend on the RNA therapeutic, the dose,
Joint with bone
the encoded protein, the carrier, and the route of administration. When
Large intestines (when relevant including
no reference can be made to published data, preliminary studies should
Peyers Patches)
be performed to assess an appropriate duration.
Larynx
9. Minimal tissue panels are available but will vary on a per-product basis.
Lymph nodes (mesenteric and any
10. Preclinical biodistribution studies do not have to comply with GLP.
peripheral)
Mammary glands 11. Preclinical biodistribution studies are not needed for vaccine products,
Oesophagus unless new administration routes, novel adjuvants or novel additives
Pancreas are used (Table 1).
Peripheral nerves
Pituitary gland
Prostate
Salivary glands (mandibular, parotid, femur or vertebrae (including bone marrow)” is required, this
sublingual) can be sternum or femur with cartilage for rodents, while the
Skeletal muscle femur may be less suited for non-rodents (due to the less uniform
Skin and subcutaneous tissue presence of active marrow and increased collection difficulty) [47].
Small intestines
Sternebrae, femur or vertebrae (including
bone marrow) 3.6. Compliance to good laboratory practice
Stomach
Thymus
Thyroid / Parathyroid glands Preclinical in vitro and in vivo pharmacology (including biodis-
Tissue masses of tumours tribution of mRNA therapeutics and mRNA vaccines) do not have
Tongue
to comply with GLP regulations [9,11,15,16,48,49]. However, when
Trachea
Urinary bladder
toxicology data is obtained alongside other information (e.g., in
Uterus with uterine cervix and oviducts preliminary dose finding studies), GLP-compliance should be
Vagina respected in the procedures which yield toxicological data
*
The EMA refers to the tissue panel for repeated dose toxicological studies and is
[9,11,49]. For example, when performing histopathology at the
therefore considerably longer. end of a non-GLP biodistribution study, every step from organ sam-
pling to the histopathological procedures should be GLP-
compliant. Drug developers can choose to perform preliminary
non-GLP-compliant toxicology studies for screening purposes.
However, GLP-compliant toxicology is a mandatory dataset for
3.5. Minimal tissue panels
the final authorization application of a chosen drug candidate.
WHO also mentions that dedicated safety pharmacology studies
The minimal panel of tissues to be examined mainly depends on
can be included in repeat dose toxicity studies, and these can
by the RNA product, the expressed protein, and the route of admin-
replace single dose toxicity studies [15].
istration. However, prespecified tissue panels have been deter-
mined and can be used as a general starting point (Table 2)
[10,11,14,46]. For example, when subcutaneous or intramuscular 4. Techniques used for authorized RNA therapeutics
injection is used, FDA requires that the draining lymph node and
contralateral site are examined as well [10]. Similarly, when Preclinical biodistribution data of therapeutics that have
inhalation is used, inclusion of tissues such as the nasal mucosa received authorization, can be found in either FDA Application
or larynx may be required. As mentioned, applicants should always Review Files (https://www.accessdata.fda.gov/scripts/cder/daf/)
consult with the regulatory agency to obtain a definitive list of tis- or the European Public Assessment Reports (https://www.ema.eu-
sues to be included in preclinical biodistribution studies. For phar- ropa.eu/en/medicines). siRNA therapeutics that have received
macokinetic studies of mRNA vaccines, no minimal tissue panel is authorization by EMA and/or FDA at the time of writing this review
available. Finally, it should be noted that the interpretation of these are OnpattroÒ (patisiran), GivlaariÒ (givosiran), OxlumoÒ (luma-
panels can be species-specific. For instance, when ‘‘Sternebrae, siran), and LeqvioÒ (inclisiran). OnpattroÒ is formulated as an
5
Table 4
RNA-containing therapeutics that received authorization by EMA/FDA.
Name Type ROA FDA-authorized EMA-authorized Techniques used

Ò
Spikevax – INN COVID-19 vaccine Moderna modRNA IM EUA Conditional QWBA
LNP-delivered bDNA assay
Ò
Comirnaty – INN COVID-19 vaccine Pfizer/BioNTech modRNA IM Yes Conditional QWBA
LNP-delivered LC-MS
BLI
LeqvioÒ – INN inclisiran naked siRNA SC Yes Yes QWBA
LC-MS
OxlumoÒ – INN lumasiran naked siRNA SC Yes Yes QWBA
LC-MS
Ò
Givlaari – INN givosiran naked siRNA SC Yes Yes QWBA
LC-MS
HPLC-probe assay
OnpattroÒ – INN patisiran siRNA IV Yes Yes QWBA
LNP-delivered LC-MS
HPLC-probe assay
ROA = route of administration, IM = intramuscular, SC = subcutaneous, IV = intravenous infusion, modRNA = nucleoside modified messenger RNA, EUA = emergency use
authorization, INN = international non-proprietary name, QWBA = quantitative whole-body autoradiography, bDNA = branched DNA, LC-MS = liquid chromatography – mass
spectrometry, BLI = in vivo bioluminescence imaging, HPLC = high-performance liquid chromatography, ‘‘naked” = not formulated as LNP.
Table 5
Radioactive isotopes used to label RNA or carrier components.
Isotope Decay type Decay energy (KeV) maximum – average half-life Epidermal penetration Range in tissue (mm) Source
3
H b 16.8–5.7 12.3 years 0.00 0.006 [59]
14
C b 156–49 5730 years 0.11 0.27 [60]
32
P b 1710–700 14.3 days 0.95 8.00 [61]
33
P b 249–76 25 days 0.35 0.60 [62]
35
S b 167–49 87.6 days 0.12 0.30 [63]
125
I* c 35 60.1 days 0.99 HVL = 20 mm [64]
KeV = kiloelectronvolt, Epidermal penetration is defined as the fraction of emitted particles that are transmitted through the human epidermis, Range in tissue = distance an
emitted particle travels in tissue, HVL = half-value layer (the amount of tissue needed to reduce the radiation intensity by 50%).
*
125I is not used to label RNA, but is included here as a reference.
LNP, while GivlaariÒ, OxlumoÒ, and LeqvioÒ are chemically modi- rated and the resulting RNA is more subject to decomposition in
fied and are conjugated to a nitrogen-containing moiety. This moi- storage [55]. All isotopes emit b particles (except for 125I, which
ety is connected to N-Acetylgalactosamine to mediate the siRNA emits gamma radiation) but the isotopes have varying half-lives
delivery into hepatocytes [50]. Like OnpattroÒ, both COVID-19 vac- and decay energies (Table 5) [56]. Compared to gamma radiation,
cines are delivered in LNPs and the mRNA COVID-19 vaccines from beta particles have lower energy and can consequently only be
Moderna and Pfizer/BioNTech are currently the only mRNA thera- detected in superficial tissues (hence the need for tissue slices).
peutics on the market. The techniques that were used to obtain However, beta-particles provide superior resolution [52]. Likewise,
33
biodistribution data for these siRNA and mRNA drugs are either P provides superior resolution compared to 32P, but is harder to
based on radiolabeling (e.g., quantitative whole-body autoradiog- produce and therefore more expensive. Other factors should be
raphy (QWBA)), liquid chromatography with online mass spec- considered as well. For example, 14C is strongly preferred over 3H
trometry (LC-MS) or hybridization assays (Table 4 and Fig. 2). because it cleaves off less readily than a 3H-carbon bond. Addition-
ally, although 3H-labeling is cheaper, easier to incorporate, and
4.1. Quantitative whole-body autoradiography and provides a better resolution than 14C, it requires a 10-fold higher
microautoradiography dose and the phosphor plates to image 3H-labeled drugs can only
be used once [52,57,58]. Contrastingly, phosphor plates for 14C-
Quantitative whole-body autoradiography (QWBA) determines imaging can be used multiple times.
the distribution of radiolabeled materials in tissues. It is considered Researchers should first determine the in vivo label stability
the industry standard for preclinical biodistribution studies and when using 3H or 125I (see the review of Solon (2012, 2015))
can be used to assess the distribution and concentration of the [52,58]. The importance of assessing the stability of the label is
RNA product itself as well as carrier components and degradation demonstrated by Christensen et al. (2013). These authors showed
products [51,52]. For example, the preclinical studies of OnpattroÒ, that 3H-labeled siRNA lost 9% of the label after 2 h and 26% after
OxlumoÒ, LeqvioÒ, GivlaariÒ and the COVID-19 vaccine of both Pfi- 48 h post-intravenous injection [65]. Nevertheless, 3H-labeling
zer/BioNTech and Moderna relied on QWBA in rats [20– was used to assess the biodistribution of GivlaariÒ siRNA. The
22,38,53,54]. Because both localization and quantification are pos- European Public Assessment Report on GivlaariÒ states that: ‘‘Other
sible, researchers can obtain tissue-specific pharmacokinetic data peaks (comprising up to 14% of total radioactivity) were shown to be
[52]. First, a radioactive isotope is incorporated in the investigated dose formulation impurities” (The report does not mention whether
compound. The isotopes that were used in authorized RNA thera- in vivo probe dissociation was examined) [54]. This may be in part
peutics are 3H and 14C, but 32P and 33P are options as well. Other attributable to probe dissociation events but is definitely worse
isotopes, like 35S and 125I are used to label peptides and proteins than the advised maximum of 3% formulation impurities (i.e., a
[52]. When using 32P, it is recommended to use labeled [a-32P] ‘‘radiopurity” of 97%) [52,58]. This can lead to over- or underesti-
UTP or [a-32P]CTP during in vitro transcription, since labeled mations of drug and metabolite quantification in different tissues
[a-32P]ATP and [a-32P]GTP are generally less efficiently incorpo- [58].
6
Fig. 1. Example of QWBA imaging, using 3H-labeling in a rat. Reprinted with permission from Bioanalysis (2015) 7(5), 557–568 as agreed by Newlands Press Ltd.
Fig. 2. Schematic overview of common labeling and detection methods for (m)RNA, proteins and LNPs. QWBA = quantitative whole body autoradiography, IHC
Ab = immunohistochemistry antibody, eGFP = enhanced green fluorescent protein, LUC = luciferase, MS = mass spectrometry, RT-qPCR = reverse transcription quantitative
polymerase chain reaction, RISC = RNA-induced silencing complex, FISH = fluorescence in situ hybridization. *IHC can also be used to demonstrate target protein knockdown
after siRNA-LNP administration.
After administration and tissue distribution of the labeled com- tions of about 20–50 mm thickness at predefined positions (de-
pounds, animals are euthanized at specified timepoints and snap- pending on the study goals) will be analyzed [51,52]. The
frozen, for example in a hexane-dry ice bath [51,52]. The frozen selected sections are freeze-dried and placed on a phosphor plate
carcasses are embedded in supporting medium and sectioned in for imaging. Quantification can be performed by including stan-
thin tissue slices [51,52,58,66]. Typically, five to ten sagittal sec- dards with a known radioactive dose and standards with a known
7
thickness. Signal acquisition usually happens inside a lead box over 4.2. Mass spectrometry-based assays
1–2 weeks. The phosphor plate can then be scanned and imaged
[51,52]. Molecular mass spectrometry (MS) identifies analytes based on
In typical QWBA studies, both albino rats (e.g., Sprague- the measured ratio between the molecular mass and the charge of
Dawley) and pigmented rats (e.g., Long-Evans) are used. The the ionized fragments. So-called tandem mass spectrometry (MS/
albino rats are included because they are also used in toxicology MS) significantly improves the confidence of identification and
studies and thus allow for better correlation between distribu- analytical resolution by fragmenting the ions in a controlled fash-
tion and toxicity. Pigmented rats are required to determine mel- ion and quantifying the characteristic molecular fragments. Fur-
anin binding, thereby predicting the radiation burden in humans thermore, coupling the mass spectrometer to an ‘‘online”
[52,58,67]. This is because melanin can bind radiolabeled com- separation step (commonly liquid chromatography (LC)) and
pounds, resulting in accumulation and a longer exposure than online detectors (e.g., fluorescence or optical absorbance) dramat-
expected [68]. QWBA can also be applied to other species, ically increases the analytical resolution. Despite the challenges
including other rodents, rabbits, dogs, pigs, and non-human pri- related to costs, skilled personnel, and its implementation in a val-
mates [52,58,69]. For example, QWBA was performed on liver idated/GLP pharmaceutical context, LC-MS/MS has become a de
samples of Cynomolgus monkeys to examine the elimination of facto gold standard for many bioanalytical chemistries due to its
LeqvioÒ siRNA [38]. unmatched combination of sensitivity, selectivity, and specificity.
QWBA can obtain tissue resolution (pixel size of about 25– Most organic molecules can be analyzed by LC-MS/MS without
100 mm2), while preserving the tissue architecture (Fig. 1). QWBA requirements for external labeling or specific functional groups
is therefore preferred over techniques that require homogenization (e.g., chromophores or fluorophores). MS-based assays are cur-
of an organ, such as LC-MS [51,52]. Moreover, the distribution data rently used to determine the presence and concentration of small
is obtained in situ, in samples that are minimally manipulated. This molecule drugs, metabolites, or carrier components in biological
bypasses exsanguination effects that inevitably occur during organ matrices (most commonly plasma). By performing serial sampling,
excision [66]. For the same reason, cross-contamination is also pharmacokinetics can be determined. Regarding RNA therapeutics,
much less of an issue compared to techniques that require excision the high molecular weights, the high anionicity, and the chemical
of organs [66]. instability of the phosphate linkages generally leads to higher
Distribution data at cellular resolution can be obtained with detection limits than are seen for most small molecules.
microautoradiography (MAR). Similar to QWBA, MAR relies on siRNAs are generally assumed to have a molecular weight
radiolabeling. Different procedures, such as the coating of micro- <15 kDa, largely making them amenable to the same analytical
scope slides with photosensitive nuclear emulsion under dark- workflows as small molecule therapeutics (with siRNA quantifica-
room conditions, making cryomicrotomic sections (4–10 mm tion limits in the low ng/ml range [77]). For example, the European
thickness), and mounting the sections under darkroom condi- Public Assessment Reports of siRNA drugs OnpattroÒ and GivlaariÒ
tions are needed for MAR. The nuclear emulsion is exposed to state that LC-MS/MS was used for quantification of siRNA in
the radioactive sections during 3 days to 2 months. Images must plasma and/or tissue samples [22,54]. The various MS modalities
be acquired in light-tight boxes at 4 °C or 20 °C (depending on for detailed analysis of oligonucleotides beyond biodistribution
which protocol is used) where the nuclear emulsion will blacken have recently been extensively reviewed [78,79] and is beyond
locally in response to the radiation [51,52,70]. Additionally, MAR the scope of this review. In contrast to siRNA, mRNA molecules
requires higher radioactive doses compared to QWBA, due to the can exhibit a molecular weight into the MDa range. Consequently,
additional magnification. Finally, the freezing process needs to MS-based quantification of an intact mRNA molecule is currently
be accelerated to prevent the formation of ice crystals that dis- not practically feasible but the recent progress in native mass spec-
rupt cellular morphology [58,66]. Researchers can use different trometry could change this in the relatively near future [80]. Up to
tissue samples from a single animal for both QWBA and MAR, now, the longest intact RNA investigated in detail (top-down
by preparing them separately. Alternatively, different animals sequencing) by mass spectrometry seems to be the 76nt tRNA in
can be used for each procedure, for example when a higher the Breuker lab [81]. Interestingly, intact viral particles, including
radioactive dose is needed for MAR, when exsanguination is per- two protein-coding RNAs of 3.1 kb and 1.4 kb, respectively, have
formed prior to MAR (which is not optimal for QWBA) [52,69], been detected by Orbitrap MS as single ion species in the MDa
or when MAR samples are chosen based on the QWBA data range [82].
[51]. Importantly, MAR is prone to user artifacts and typically Biodistribution analysis of RNA therapeutics adds significant
does not permit reliable quantification [52]. Preclinical studies challenges as compared to quantification and structure elucidation
for OnpattroÒ most likely used MAR, as they contain data on of neat RNA. Indeed, RNA therapeutics in a biological matrix con-
intracellular radioactivity in hepatocytes, hepatic vascular cells tains high concentrations of endogenous RNA, as well as proteins
(cell type not specified), and liver sinusoid lumen [22]. Obtaining and other biomolecules that can form strong interactions with
cellular resolution has a more prominent role when cell- RNA. MS-based RNA analysis (or their metabolites) then becomes
targeting moieties are used on LNPs, for example [71–76]. In this a challenge of robust and efficient sample preparation and analyte
regard, MAR can sometimes (depending on reagent compatibil- extraction prior to analysis (e.g., through trizol extraction, protein
ity) be coupled to immunohistochemistry to verify targeted digestion, solid-phase/bead-based extraction, and online trapping
delivery [52]. columns) [83].
The use of radioactive substances requires special handling and MS-based detection of siRNA in biological samples usually does
dedicated infrastructure. In addition, reliable quantification not require modifications or labeling methods. Indeed, most ther-
requires extensive training and/or experience, and the required apeutic siRNAs incorporate one or more non-endogenous chemical
equipment is very expensive [52,58]. Consequently, QWBA and modifications to enhance stability or therapeutic effect [38,53,54].
MAR are typically outsourced. Using QWBA or MAR, it is impossi- This provides very good signal-to-noise ratios (SNR) and low detec-
ble to distinguish between the drug (or carrier) itself, its metabo- tion limits in LC-MS/MS after forced depolymerization (unpub-
lites, or its degradation products [52,58,66]. However, QWBA is lished data from our laboratories at SINTEF). The same principle
non-destructive and can therefore be used in tandem with other would apply to mRNA therapeutics if one or more incorporated
techniques (e.g., LC-MS) to further characterize the obtained nucleotides are non-endogenous. An interesting approach would
radioactive signals [51,52]. be to incorporate stable (non-radioactive) isotope-labeled nucleo-
8
tides in the mRNA. For example, an isotope-labeled 50 cap would cause an unwanted background signal (Section 5.2) [96,109,113],
generate non-endogenous heavy-labeled (typically 2H, 13C, 15N) that FISH is time-consuming and requires some experience, and
monomers upon nuclease digestion. Such stable isotope analogues, that the necessary fixation prevents using the sample in other
widely used in MS-based small molecule- and proteomics studies, downstream assays [96,114].
are chemically indistinguishable from their non-labeled counter- The preclinical studies for GivlaariÒ and OnpattroÒ utilized
parts yet easily discernible in MS. The limiting factor of this hybridization techniques in tandem with high-performance liquid
approach would be the cost of the stable isotope nucleotides. The- chromatography (HPLC) [22,54]. A fluorescently labeled dye was
oretically, MS-based detection of radioactive isotopes (as used in attached to a synthetic oligonucleotide, complementary to the tar-
QWBA and MAR) is also possible but would require sufficient ded- get siRNA. After hybridization, HPLC was performed to isolate
icated facilities. It was recently shown [84] that a non-endogenous hybridized probes from e.g., unbound probes. Finally, fluorescence
phosphorothioate fragment of an antisense oligonucleotide could was used to quantify the siRNA.
be used as a marker for biodistribution in matrix assisted laser Branched DNA (bDNA) amplification relies on a series of inter-
desorption/ionization-Fourier transform-ion cyclotron resonance- connecting DNA probes which amplify a fluorescent signal after
MS (MALDI-FT-ICR-MS) imaging. The use of MALDI ionization for binding the target RNA [93]. The signal amplification is linear
direct on-tissue, spatially resolved quantification for therapeutics and therefore allows reliable quantification [115] and even allows
has brought exciting results for small molecule drugs [85] and even for multiplexing [116]. A multiplexed bDNA assay was used in the
antibodies [86], and it remains to be seen if this applicability can be preclinical studies of Moderna’s COVID-19 vaccine to simultane-
extended fully to RNA therapeutics. This would, however, provide a ously examine the biodistribution of 6 mRNAs [21]. Although ini-
significant improvement, as sample manipulation is drastically tially developed for detecting nucleic acids in solution, bDNA
reduced, thereby avoiding cross-contamination and exsanguina- amplification has been used on frozen tissue samples mounted
tion effects [52,58]. Moreover, because organ homogenization is on slides [117].
not needed, MALDI also provides spatial information [58].
Most RNA therapeutics are currently delivered in LNPs contain-
5. Other techniques that could reach EMA/FDA requirements
ing one or more non-endogenous lipids, often with intramolecular
groups that ionize very well. This provides very good detection
QWBA is the gold-standard for preclinical biodistribution stud-
limits in LC-MS/MS. For example, the European Public Assessment
ies. However, this technique has some critical limitations for RNA
Reports of OnpattroÒ and the Pfizer/BioNTech COVID-19 vaccine
therapeutics and especially for mRNA therapeutics. Among these
state that LC-MS/MS was used to assess the pharmacokinetics
limitations are the fact that the expressed protein is not tracked
and biodistribution of the synthetic lipid components [20,22].
and the inability to discriminate metabolites and degradation
The same assay methodology was used to optimize the pharma-
products. The techniques described in this chapter (Fig. 2) avoid
cokinetics of several LNP formulations of Moderna [87]. The biodis-
the use of radioactive materials. However, they have the common
tribution of LNPs can also be used to approximate the
downside that they are currently not thoroughly validated and that
biodistribution of the RNA payloads [88]. In addition, MS-based
they often require additional processing steps.
targeted proteomics approaches can help detect elevated thera-
peutic protein concentrations after mRNA translation.
5.1. Reverse transcription quantitative polymerase chain reaction
4.3. Hybridization techniques
Although the EMA and the FDA recommend RT-qPCR for biodis-
Fluorescence in situ hybridization (FISH) is the gold standard for tribution studies, the exact criteria for performing these studies are
single-molecule RNA visualization on fixed samples and is not yet defined. In addition, no acceptance criteria for assay valida-
described for both siRNA [89–92] and mRNA [93–104]. Detection tion have been determined. Experts in the field recommend testing
of single RNA molecules is typically achieved by hybridizing mul- 3 primer pairs and using probe-based qPCR analysis over DNA-
tiple fluorescently labeled probes on the target RNA [93,105,106]. binding fluorescent dyes due to their superior specificity and pos-
As a possible advantage over QWBA, probes targeting multiple sibility to multiplex (the added cost of probes is compensated by
regions on the mRNA can be labeled with different fluorochromes fewer labor hours on method development) [118]. Evidently,
thereby providing information on the integrity of the target mRNA. PCR-based methods require careful sample extraction from tissue
Moreover, different target mRNAs (e.g., in multivalent mRNA vac- homogenates and bodily fluids to avoid sample cross-
cines) can be visualized simultaneously [99,100,107–109]. The contamination. Sample collection is typically performed in
additive nature of FISH makes it an interesting technique that ‘‘RNAlater” reagent to minimize RNA degradation, which can be
could be used in conjunction with other imaging modalities such evaluated by e.g., quantifying host housekeeping genes or by cap-
as in vivo bioluminescence imaging (BLI) or in vivo fluorescence illary gel electrophoresis. Each 96-well plate should include appro-
imaging (FLI). For example, a protocol was published which priate quality controls and a 10-fold dilution series of target RNA to
enables the ex vivo imaging and colocalization of endogenous allow absolute quantification over a wide dynamic range. These
mRNA (through FISH) and transgenic eGFP on the same hippocam- controls also indicate the efficiency and accuracy of the reverse
pal tissue section [110]. Moreover, selective probes can be transcription reaction and should be evaluated in the presence of
designed for codon optimized synthetic mRNAs (even when encod- total RNA from negative control samples. Although one-step RT-
ing endogenous proteins). Similarly, high-fidelity FISH allows for qPCR minimizes the risk of cross-contamination and technical
detection of single-nucleotide variation in target mRNA [111]. errors, the need for reverse transcription always introduces vari-
The broad variety of available fluorescent compounds also allow ability and should therefore be carefully interpreted [118].
co-staining with structural or functional markers for cellular iden- An RNA biodistribution assay is expected to have a lower limit
tification and even subcellular localization. Interestingly, FISH can of quantification of less than 50 copies per mg of input RNA [118].
also be combined with immunohistochemistry to visualize both However, small quantities of target RNA can sometimes be masked
the mRNA and its translated protein [93,112] or (sub)cellular colo- by large quantities of endogenous RNA. To address this problem,
calization markers. Importantly, quantification of FISH data conventional qPCR can be replaced by digital droplet PCR (ddPCR)
requires software that must be validated. Finally, it should be which partitions a particular sample into many small droplets
noted that that autofluorescence or aberrant probe-binding can where individual PCR reactions occur. The number of positive over
9
Table 6 mRNA remains encapsulated in LNPs or never escapes the endo-

A comparative summary of FLI and BLI. somes [120]. This could result in an over-estimation of synthetic
In vivo fluorescence imaging (FLI) In vivo bioluminescence imaging RNA in tissues. Especially for protein replacement therapies, PCR-
(BLI) based detection might even be unable to discriminate between
Light source exogenous and endogenous mRNA, although careful codon selec-
Light at a particular wavelength A chemical substrate is enzymatically tion could help address this issue.
excites fluorophores which in turn converted. This reaction also
emit light of a longer wavelength produces light.
5.2. Fluorescence and bioluminescence
Pros
Wide variety of excitation/emission High SNR (minimal photon
combinations, facilitating production in the absence of
In vivo fluorescence imaging (FLI) and in vivo bioluminescence
multicolor imaging substrate) imaging (BLI) are two very common imaging modalities (Table 6).
High spatial and temporal resolution No phototoxicity or physiological In the case of FLI, light at a particular wavelength excites fluoro-
responses due to excitation light, no genic dyes or proteins which in turn emit light of a longer wave-
photobleaching
length. In contrast, BLI is based on an enzymatic reaction that
Any light source can excite Growing range of luciferase enzymes
fluorophores, thereby facilitating with different substrate generates light while converting a chemical substrate [121].
tandem dyes, FRET, BRET, etc. combinations (e.g., requiring various BLI and FLI require photons to pass through an animal or tissue
cofactors thereby serving as sample before reaching a detector. These tissues absorb and scatter
biosensors) light primarily at wavelengths below 600 nm and above 900 nm
Glow and flash luciferases available
(Optical window, Fig. 3a), This has two important consequences.
to match experimental needs
First, both excitation light and emission light get absorbed and
Cons
Sometimes high background due to Long-term imaging requires
scattered, which results in dimmer images as the tissue thickness
tissue-autofluorescence (reduces continuous substrate increases [122]. The absorption of high-intensity excitation light
SNR) supplementation and substrate can also result in phototoxicity, especially with repeated exposure
kinetics need to be considered [121]. Secondly, naturally occurring fluorogenic compounds also
Absorption of light by water, Relatively dim compared to FLI
produce light that coincides with the emission of the fluorochrome
hemoglobin, melanin etc. (about 1:100), requiring longer
(reducing SNR). Using nude, exposure times thereby limiting under investigation. BLI does not require high intensity excitation
shaved or albino mice can help temporal resolution. When binning is light and its dim emission light causes minimal tissue fluorescence
mitigate this effect. required, spatial resolution is also [121]. Contrastingly, the strong light required to excite fluo-
limited rophores typically causes autofluorescence in the blue-green spec-
Phototoxicity can hamper sequential Broad emission spectra thereby
trum [123].
imaging limiting multicolor imaging. In
addition, spectral unmixing is only To minimize absorption, phototoxicity, and tissue fluorescence,
possible on the emission spectrum emission in the near-infrared (NIR, wavelength 700–900 nm) can
Excitation light can cause photon- No optical sectioning, causing blurry be used (Fig. 3b) [124]. Note that a second NIR-window exists
induced, unwanted physiological images when thick samples are used
(NIR-II, wavelength 1000–1700 nm), that can also be used for
responses
Photobleaching
in vivo imaging [125]. Additionally, spectral unmixing can help fil-
ter out the tissue fluorescence. Background fluorescence from diet-
Required equipment
Excitation light source with series of Dedicated luminescence imager and ary components (e.g., chlorophyll) in the gut can be minimized by
filters. Camera with emission software. A fluorescence microscope feeding the rodents with low-fluorescent diets [126,127]. In mice,
filters. is often not sensitive enough due to the effect of a diet change is visible within 1–2 days, but the intake
inefficient transmission of light of feces can prolong this period. In practice, it is therefore advised
Many compatible machines and Appropriate substrate and cofactors
to combine the dietary change with a cage change or to extend the
setups such as flow cytometers,
(confocal) fluorescence waiting period [126]. As mentioned, melanin can contribute signif-
microscopes, FLI systems, multi- icantly to the absorption of light (Fig. 3a). Using albino mice, nude
photon excitation mice, or shaved mice should therefore be considered as well [128].
Most fit for Finally, FLI and BLI are not mutually exclusive and can be used
Non-living samples, multiplexing, Long-term or repetitive imaging of in parallel [129], as fusion proteins [130], and as BRET partners
very high spatial and/or temporal live samples (e.g., low-abundance
(Section 5.2.2).
resolution proteins or fast dynamics), photo-
sensitive samples
5.2.1. In vivo bioluminescence imaging
SNR = signal-to-noise ratio, FRET = Förster or fluorescence resonance energy BLI is characterized by a very high SNR and does not require
transfer, BRET = bioluminescence resonance energy transfer.
excitation by an external light source. Prolonged imaging is easily
achievable, but substrate kinetics should be taken into considera-
tion. Regarding preclinical biodistribution studies, BLI can be used
negative droplets is then analyzed via Poisson distribution to to characterize the translation kinetics of mRNA therapeutics. By
determine the target RNA concentration. Benefits of ddPCR include exchanging the coding sequence of the therapeutic protein with
improved sensitivity, precision, and reproducibility. However, the sequence of a luciferase, a luminescence signal can be acquired
preparation and analysis of ddPCR samples takes approximately that is proportional to the amount of protein produced. This cannot
7 times longer than conventional qPCR and often involves more be achieved with QWBA and is particularly useful to evaluate the
method optimization [118]. distribution and performance of a new mRNA carrier [131–139].
RT-qPCR could be an important tool in determining tissue The argument that the sequence of the mRNA is unlikely to have
biodistribution of RNA therapeutics. It is however currently a meaningful effect on the mRNA-LNP biodistribution was used
unclear to what extent this technique discriminates between intact for the Pfizer/BioNTech COVID-19 vaccine [20]. After intramuscular
and degraded RNA [119]. Moreover, there are no publications administration to mice, in vivo bioluminescence was determined
available which utilize this technique in an RNA biodistribution using an In Vivo Imaging System (IVIS) at six time points (i.e.,
context at the time of writing. In addition, not all amplified mRNA 6 h, 24 h, 48 h, 72 h, 6d, 9d post-injection) [20]. These longitudinal
necessarily derives from the intracellular compartment as most measurements were performed in only 6 anesthetized mice. Addi-
10
Fig. 3. (a) Absorption spectra of water, melanin, and oxyhemoglobin. Absorption is minimal in the optical window (white section in both (a) and (b)). (b) Emission spectra of
eGFP, miRFP, Cy7, Firefly luciferase (Fluc), Renilla luciferase (hRluc), and NanoLuc (Nluc).
tionally, hepatic delivery efficiency of siRNA with LNPs can be exposure times (seconds to minutes) [149]. Moreover, the
examined by evaluating knock-down of luciferase mRNA observed broader distribution in the QWBA assay can also be
expressed in the liver of mice [140]. attributed to radiolabeled degradation products or metabolites,
A broad range of luciferases are available and are discussed in especially since unstable 3H-labeling was used [52,57,58,66].
detail elsewhere [121,124,141–145]. Different luciferases require The limited spatial resolution of BLI is an important drawback
different cofactors (e.g., ATP, O2, or Mg2+) and substrates with, for in biodistribution studies. Moreover, the relative dimness of BLI
example, increased brightness or red-shifted emission spectrum. often necessitates ‘‘binning”. This methodology combines the sig-
The kinetics of the substrate-dependent chemical reaction is nal of multiple adjacent pixels into one exponentially brighter sig-
another important factor. Some luciferases (e.g., Firefly luciferase nal. Unfortunately, binning results in a further reduction of spatial
(Fluc)) are characterized by light emission that steadily increases resolution. Possible workarounds for the lower spatial resolution
before reaching a plateau phase (where quantification should be include decreasing the distance between the luminescent light
performed). This kinetic profile is referred to as ‘‘glow” kinetics. source and the detector (which are exponentially related). Another
Other luciferases (such as Renilla luciferase (Rluc) and Gaussia workaround is to image individual organs ex vivo. However, this
luciferase (Gluc)) will convert the substrate much quicker, reach- requires additional tissue handling, which can then influence
ing a higher peak luminescence within seconds and rapidly decay- quantification (e.g., due to variable exsanguination) and introduce
ing afterward. Quantification of these ‘‘flash” kinetics should be cross-contamination. Alternatively, BLI can be used to sensitively
performed immediately after the substrate is provided and are detect signal in a crudely demarcated region, followed by a second
much more prone to technical variability. In practice, glow type technique (e.g., FLI) to determine the exact location of the signal
kinetics are preferential in vivo, while the brighter flash kinetics origin. BLI assays can also be followed-up with, for example, flow
of RLuc can be beneficial in vitro. Apart from the luciferase itself, cytometry and immunohistochemistry based on anti-luciferase
the substrate can also have an influence on the reaction kinetics. antibodies. Finally, more accurate 3D spatial information can be
The recently developed NanoLuc is based on bright flash kinetics obtained by using BLI tomography [150,151].
(reported to be 150-fold higher than that of Fluc) that can be sta-
bilized to mimic glow kinetics [146]. This enzyme and the accom- 5.2.2. In vivo fluorescence imaging
panying substrate have been commercialized by Promega LNPs can be fluorescently labeled with lipophilic tracers (e.g.,
(Fitchburg, WI, US). Unfortunately, the wavelength of the emitted DiD, DiR) [137,152–154], but this can potentially influence their
light is relatively short and therefore not yet optimal for in vivo physicochemical properties and biodistribution. Additionally, lipo-
use (Fig. 3). Finally, it should be noted that no apparent toxicity philic tracers can leach out the LNPs. Therefore, this approach is
has been reported for the substrates of, Firefly or Renilla luciferase, not optimal for biodistribution studies as regulatory agencies
apart from skin and mucosal irritation [147,148]. might reject the data [8,9,11,14]. Fortunately, the coding sequence
Despite the excellent SNR, BLI is known to be relatively dim of mRNA therapeutics or vaccines can be replaced without signifi-
[121]. This is also mentioned in the preclinical studies of the Pfi- cantly influencing mRNA-LNP distribution (Section 5.2.1) [20]. By
zer/BioNTech COVID-19 vaccine. When the same luciferase mod- introducing the coding sequence of a fluorescent protein (e.g.,
RNA containing LNP was radiolabeled and re-examined in rats, a eGFP, miRFP) [133,155] or by fusing the therapeutic/antigenic pro-
broader biodistribution was determined. Although this was attrib- tein to a dye-binding tag (e.g., HaloTag) [156], the distribution of
uted to the higher sensitivity of the QWBA assay, it is important to an mRNA-LNP and its translation kinetics can be examined. Addi-
note that both techniques were not compared directly. There were tionally, fluorescence/Förster resonance energy transfer (FRET)
many differences between both studies, including animal species can also be used, for example, by including a FRET fusion protein
and administered dose. Moreover, QWBA is determined on sagittal in the coding sequence of mRNA. In a second example, labeled
tissue sections, while BLI images are acquired in whole animals siRNA and nanoparticles formed a FRET-pair. Once the siRNA
thereby significantly diminishing and scattering the signal (but escape the confines of the LNP, fluorescence could no longer occur
providing longitudinal data in the same animal). In addition, [157]. FRET increases the total brightness and/or shifts the emis-
QWBA exposure times (days to weeks) are much longer than BLI sion spectrum to NIR by using the emission from a primary fluores-
11
cent protein to excite a second fluorescent protein [155]. Similarly, should have minimal impact on the biodistribution or on the trans-
bioluminescence resonance energy transfer (BRET) uses a lucifer- lation dynamics of the mRNA but can potentially influence mRNA
ase to excite an adjacent fluorescent protein stability [171]. These aptamers selectively bind fluorescent dyes
[121,124,141,142,155]. Consequently, BRET does not require exter- but currently lack the ability to visualize single mRNA molecules
nal excitation light, thereby significantly reducing tissue autofluo- [93,171]. Background fluorescence by unbound dyes can be
rescence and potential phototoxicity. reduced, by using aptamer-binding fluorophore-quencher pairs
To limit the number of animals needed, developers can co- which only become fluorescent when bound to the aptamer
encapsulate fluorescent protein-encoding mRNA and luciferase- [93,171]. Alternatively, unique protein binding motifs (e.g., MS2,
encoding mRNA in a single LNP, which mimics multivalent mRNA PP7, kN) can be introduced in the 30 UTR of mRNA. These motifs
vaccines [158,159]. Alternatively, it is also possible to encode an interact with RNA-binding proteins that are fused to fluorescent
eGFP-Fluc fusion protein on a single mRNA instead [130]. proteins and allow visualization of single mRNA molecules
mRNA and siRNA can also be labeled directly. This approach is [93,171]. Another interesting system to visualize single RNA mole-
however accompanied by some challenges. For example, cyanine cules is based on a catalytically inactivated Cas13 protein (fused to
dyes such as Cy5 and Cy7 (Fig. 3b) can be chemically linked to a fluorescent protein). When supplied with a custom-made guide
RNA molecules [160–163], but these bulky and hydrophobic RNA, the Cas13 will sequence-specifically bind a target mRNA of
groups can significantly interfere with translation dynamics choice [171]. Finally, molecular beacons are oligonucleotides that
[164]. Other dyes, such as the more hydrophilic Alexa FluorÒ 647 contain a fluorophore on one end of a stem loop, and a quencher
and 750, have also been used to label siRNA [120,165]. The label on the other end. The stem loop opens upon binding its target
can then be imaged in vivo using fluorescence tomography, cou- RNA, elevating the influence of the quencher [93,171].
pled to micro-CT [165]. Alternatively, the organs can also be
imaged ex vivo [166]. Other labeling approaches include the intro-
duction of fluorescent nucleosides, 50 -cap, 30 -polyA tail, or even 6. Final remarks
introducing dye-binding aptamers [164]. Unfortunately, these
approaches either introduce insufficient labels for in vivo imaging, Biodistribution studies intend to gain insight into the where-
or they still perturb transcription and/or translation [164]. An abouts of injected drugs. This knowledge is then used to help inter-
interesting direct RNA labeling solution was recently developed pret the drug’s pharmacological or toxicological interactions.
by Baladi et al. (2021) [164]. These authors used a fluorescent tri- Interpreting all these interactions is a daunting task for mRNA
cyclic cytosine analogue, which can comprise up to 100% of the therapeutics since these novel medicines contain many compo-
cytosines in a 1.2 kb-long GFP-encoding mRNA [164]. Unlike other nents and are processed on multiple levels. For example, PEGylated
labeling methods, direct incorporation of the fluorescent cytosine LNPs can potentially elicit immune responses [28], LNPs can tem-
analogue had minimal influence on transcription and translation porarily saturate the scavenging systems in the liver [172], impu-
kinetics [164]. Evidently, this labeling method still requires further rities such as dsRNA can trigger the production of pro-
examination and validation before use in preclinical biodistribu- inflammatory cytokines and cause antiviral states in cells [30,31],
tion studies. therapeutic mRNA can act as miRNA sponges [173], and the
An important aspect is the in vivo stability of a fluorescent label. expressed therapeutic protein can have local or even distant effects
This is the main reason that hybridization probes, such as FISH in the body. In addition, current regulations for preclinical biodis-
probes cannot be used in vivo [164]. Still, Kirschman et al. (2017) tribution data of (m)RNA therapeutics are vague and ill-defined
have used a multi-labeled probe and a handheld NIR fluorescence without concrete specification on e.g., thresholds for sensitivity.
camera to successfully track intramuscularly injected mRNA in vivo Evidently, expanding the clinical applications and public accep-
[106]. The in vivo probe-mRNA interaction was verified 2 h after tance of this very promising platform technology would greatly
injection through a co-localization FISH assay [106]. This time- benefit from a more robust regulatory framework. Pivoting from
frame is however not yet long enough for longitudinal mRNA dis- a per-product approach to more general guidelines may become
tribution studies. A similar attempt to design an RNA binding a necessity, as the number of (m)RNA therapeutics applying for
probe was recently published by Wu et al. (2020), where a tripar- clinical approval increases rapidly.
tite DNA probe was injected intravenously and intratumoral to mRNA vaccines against infectious diseases are currently not
image a micro-RNA target [167]. Unfortunately, the current probe considered as gene therapeutics by regulatory agencies such as
design resulted in very limited spatial resolution in vivo. EMA and FDA. Instead, they are regarded as vaccines despite their
Not all fluorescently labeled RNA is directly accessible for trans- identical composition and production process as mRNA therapeu-
lation (e.g., in LNPs, phagosomes, or endosomes [120]). To monitor tics for protein replacement. Indeed, many mRNA-based therapeu-
translatable mRNA, Ai14 reporter mice can be used. This mice tics (including mRNA vaccines) rely on recombinant DNA
strain carries a fluorescent protein transgene (e.g., tdTomato) of technology for in vitro transcription template production. Based
which the transcription is inhibited due to an upstream LoxP- on this property, almost all mRNA-based therapeutics should be
flanked stop cassette. When Cre recombinase mRNA is translated classified as ‘‘gene therapy medicinal products”, as defined by the
in these mice, the stop cassette is removed and the cells become EMA [174] (of note, this does not apply to siRNA-based therapeu-
permanently fluorescent through expression of the tdTomato pro- tics as these molecules are typically chemically manufactured).
tein [168–170]. This reporter model has been used to investigate Additionally, mRNA vaccines against non-infectious diseases such
tissue distribution and cytosolic mRNA delivery of altered LNPs as cancer are not regarded as mRNA vaccines, but as gene therapy
[168,169]. Of note, this version of the system is less suitable for products. Strikingly, this means that an mRNA vaccine against
evaluation of expression dynamics, as the fluorescence observed human papilloma virus (HPV)-induced malignancies is classified
is not expected to correlate well to the amount of Cre recombinase as a gene therapy, whereas using the same mRNA for HPV vaccina-
translated. tion classifies it as a vaccine [174]. The rationale (and implications)
Lastly, additional molecular imaging tools have been designed for this distinction is unclear but may rely on the added effect of
for cell cultures and can therefore also be used to image target adjuvants and the antigenic nature of the translated exogenous
mRNA on tissue sections obtained during biodistribution studies. proteins of mRNA vaccines. Although these exogenous proteins
These techniques include aptamers (e.g., Spinach, Broccoli, and are unlikely to have a physiological function in the body, these vac-
Mango) in the 30 untranslated region (30 UTR) of an mRNA, which cines are meant to elicit robust long-lasting immune responses and
12
are therefore not biologically inert. Moreover, tissue biodistribu- References

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Declaration of Competing Interest Use, EPAR Comirnaty, INN-COVID-19 mRNA Vaccine (nucleoside-modified)
(EMA/707383/2020 Corr.1)), 2021. https://www.ema.europa.eu/
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The authors declare that they have no known competing finan- en.pdf.
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to influence the work reported in this paper. Use, EPAR COVID-19 Vaccine Moderna, INN-COVID-19 mRNA Vaccine
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Acknowledgements [22] European Medicines Agency - Committee for Medicinal Products for Human
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Regulatory Guildilens For Preclinical Tools To Study Biodistibution of RNA Therapeutucs

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Advanced Drug Delivery Reviews 184 (2022) 114236

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Regulatory guidelines and preclinical tools to study the biodistribution

1. Introduction Consequently, this FDA guidance document contains the most

Europe and United States

Name Type ROA FDA-authorized EMA-authorized Techniques used

Table 6 mRNA remains encapsulated in LNPs or never escapes the endo-

are therefore not biologically inert. Moreover, tissue biodistribu- References

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