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Chapter 10
https://t.me/med1917
Drug Design Models forHuman Diseases
by Transgenic Animals
SabyasachiBanerjee, SubhasisBanerjee, SankhadipBose, AvikDas,
SantanuBanerjee, andRajendraGyawali
Abstract The procedures of drug research and preclinical trials are difcult, and
a signicant majority of medication candidates are unable to receive authorization from the United States Food and Drug Administration. To enhance the likelihood of success in the new drug development process, it is necessary to use
efcient and predictive techniques that can identify high-quality targets. An
effective approach to address the difculties encountered in the advancement of
novel medications and combination treatments is using cost-effective and easily
controllable animal models for invivo experimentation. The use of transgenic
animals in the drug discovery laboratory as a model is considered an important
tool for researching human diseases and determining responses to therapeutic
intervention. These animals are genetically modied organisms that exhibit characteristics resembling symptoms of certain human illnesses. It offers genetic
models of many human illnesses that are crucial for comprehending diseases and
establishing novel targets. Since the 1980s, scientists have achieved the ability to
manipulate the mouse genome via genetic modication, namely by eliminating or
replacing a particular gene. ATryn, the rst medication derived from transgenic
S. Banerjee (*) · S. Banerjee
Department of Pharmaceutical Chemistry, Gupta College of Technological Sciences,
Asansol, West Bengal, India
S. Bose
School of Pharmacy, The Neotia University, Sarisa, West Bengal, India
A. Das · S. Banerjee
Department of Pharmacology, Gupta College of Technological Sciences,
Asansol, West Bengal, India
R. Gyawali
Department of Pharmacy, Kathmandu University, Dhulikhel, Nepal
Ltd. 2024
S. Bose et al. (eds.), Concepts in Pharmaceutical Biotechnology and Drug
Development, Interdisciplinary Biotechnological Advances,
https://doi.org/10.1007/978-981-97-1148-2_10
189© The Author(s), under exclusive license to Springer Nature Singapore Pte
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animals, was produced and authorized by the United States Food and Drug
Administration within 20years of its creation. This breakthrough has paved the
way for the development of pharmaceuticals derived from transgenic animals.
One such application is the creation of biologically safe medications that are
based on human regulatory proteins. Many issues, such as ethical concerns, regulatory requirements, and patents pertaining to the usage of transgenic animals,
will get additional attention in the coming years. Transgenic animals play a critical role in drug discovery and development with potential applications in xenotransplantation, clinical trials, and other areas.
Keywords Transgenic animals · Genetically engineered mice · Models · Drug
development · Drug design
S. Banerjee et al.
10.1 Introduction
The development of a new marketable drug product is becoming costly because of
the biological complexities of human diseases, research and development process,
preclinical and clinical studies, compound manufacturing process, regulatory policy, and prolonged time for market entry. The attrition rate of these procedures is
substantial, with fewer than 10% of the substances examined in clinical trials receiving clearance from the United States Food and Drug Administration (US FDA)
(Zambrowicz and Sands 2003; Sharpless and DePinho 2006). The expenditure associated with introducing a novel pharmaceutical surpasses $1 billion. Hence, it is
crucial to have technical advancements that enable convenient and effective identication and validation of targets in the rst stages of drug development. This is
necessary in order to decrease the risk of failure and the expenses associated with
introducing a new medication (Begley and Ellis 2012). Utilizing appropriate methodologies that simulate the behavior and forecast the effectiveness of medication
would enhance the overall rate of success and provide a consistent approach to the
advancement of drugs.
An effective approach to address the signicant obstacles encountered in the
development of novel medications and combination treatments is using inexpensive
and easily controllable animal models invivo (Hansen and Khanna 2004; Suggitt
and Bibby 2005). For drug testing, wild-type or spontaneously altered mice were
the only options until 1980. The animal model systems were crucial in the development of several chemotherapeutic drugs, such as alkylating and other DNAdamaging compounds, that are now in use (Esteller etal. 2000). Immunocompromised
mice were recently used to evaluate potential anticancer drugs (Sharpless and
DePinho 2006; Suggitt and Bibby 2005). Nevertheless, there was signicant
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variation in the ndings of these screens when comparing the reactions in mice and
humans. These models demonstrated limited capability in forecasting the outcomes
of clinical trial Phase I or II (Richmond and Su 2008).
Genetically engineered mouse models (GEMMs) of human ailments have the
potential to enhance medication development (Politi and Pao 2011; Kucherlapati
2012). Various kinds of GEMMs, including transgenic, knock-in, and knockout
mouse models, have been used in drug research and preclinical studies since the
1980s (Politi and Pao 2011; Frese and Tuveson 2007). Transgenic innovation
enables the integration of additional DNA that contains the desired gene into the
mice genome. Knock-in or knockout technology allows for the targeted removal or
modication of specic sections of the mice genome.
The advancement of new transgenic techniques provides better and more genetically altered animals and is employed in a broad range of biological, pharmaceutical, and safety testing scenarios. The genomes of three widely studied organisms—the
human, rat, and mouse have been deciphered, speeding up this development
(Houdebine 2009). Pharmaceutical businesses encounter the obstacle that around
10% of substances examined in clinical trials successfully reach the marketplace,
and among those, only a small portion will provide substantial prots. The expense
associated with discovering novel drugs is substantial, amounting to around $800
million in the United States. Of this amount, 80% is allocated toward clinical trials
and improvement. There is hope that transgenic technology might reduce drug
development failures by improving the quality of drug targets and compounds
(Snaith and Törnell 2002).
In the last three decades, the methods for modifying the genetic code of mice and
their embryos have advanced signicantly. For instance, researchers have constructed inducible and conditional models that allow them to temporarily activate or
deactivate genes, either in specic locations or for a certain period of time (Hansen
and Khanna 2004; Frese and Tuveson 2007; Sun et al. 2007). Knock-in models
include the insertion of transgenes at a specic location or the introduction of specic mutations into target genes. The advanced mouse models created via these
advancements in mice engineering approaches are valuable for drug development
and preclinical studies.
In recent years, GEMMs have become an indispensable tool for determining
target function, verifying selectivity, and making predictions about toxicology
and drug development. Over the last three decades, researchers have created
several mouse models (Kucherlapati 2012; Frese and Tuveson 2007). The primary roles of GEMMs in drug development are target conrmation, pharmacodynamic indications of drug action identication, toxicity identication, and
safety assessment (Politi and Pao 2011) (Table10.1). GEMMs have also been
used to verify assumptions about the plausibility of a novel gene as a possible
therapeutic target.
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Table 10.1 Applications of GEMMs in drug development (Lee 2014)
Drugs GEM phenotype Target gene
Evista, Premarin Knockout mice exhibited impaired fertility and
decreased bone mineral densities
Epogen, Procrit Individuals with a targeted null mutation in both sets
of the gene show a decrease in the production of
early red blood cells and do not survive beyond
embryonic day 13 owing to a disruption in the
formation of red blood cells in the fetal liver
Zyrtec, Allegra,
Claritin
Celebrex Knockout mice exhibited diminished inammation,
Celexa, Effexor,
Zoloft, Paxil,
Prozac
Knockout mice exhibited reduced B- and T-cell
reactivity, decreased vigilance, and modied activity
levels
a signicant decrease in collagen-induced arthritis,
reduced fever response, and reduced production of
polyps
Individuals with a targeted null mutation in a
homozygous state have signicantly reduced
amounts of serotonin in the brain and demonstrate
increased levels of anxiety
S. Banerjee et al.
Estrogen Esr1, Esr2
receptors
Erythropoietin
Histamine H1
receptor
Cox2
Serotonin transporter
(5-HTT)
10.2 Preclinical Trials andDrug Development Using New
Genetically Modied Mouse Models
Although GEMMs have signicant benets in drug design and preclinical examinations, there are certain technological constraints that hinder the creation of new
GEMMs (Beard etal. 2006). Transgenic mice exhibit signicant variability among
different individuals, whereas knockout mice may not always display the anticipated phenotypes and may sometimes exhibit entirely new or unexpected traits.
Another signicant issue is the duration of generation for GEMMs. The timescales
necessary for the production of transgenic and knockout mice are very lengthy,
spanning from 12 to 24months from the original design phase to the formation of
the rst experimental group. Several researchers interested in genetically engineering mice have explored novel approaches to altering the mouse genome and embryos
in order to create and utilize GEMMs. Targeted transgenesis is a method that
decreases the expense and speeds up the process of generating GEMMs (Beard
etal. 2006; Seibler etal. 2005). This method further enables the transgene’s longterm incorporation and expression in the mouse genome. RNA interference (RNAi)
and inducible transgenic mouse models are two examples of the new types of mice
made possible by targeted transgenesis. These models allow for the regulation of
specic genes, either by knocking them down or inducing their expression, in a
temporal or spatial manner (Lewandoski 2001; Kleinhammer etal. 2011). These
models are expected to boost the usefulness of GEMMs in drug discovery and open
up new avenues for their use. The generation of GEMMs may be streamlined and
made more efcient by targeted transgenesis. These approaches will be used to create many transgenic and RNAi mouse models.
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10.2.1 Advantages andDisadvantages ofGenerating GEMMs
Using Engineered Nucleases
Engineered nucleases provide new approaches for creating GEMMs. Currently,
there are three types of engineered nucleases that have been created: transcription
activator-like effector (TALEN) nuclease, zinc-nger nuclease (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated
(Cas) system (Gaj et al. 2013). These techniques enable the production of live
knock-in or knockout mice without the need for chimeras, resulting in time and cost
savings compared to traditional procedures that use Embryonic stem (ES) cells and
homologous recombination.
There are other concerns that need to be thoroughly evaluated before producing
and using these mice with the use of modied nuclease. First, a signicant problem
arises from the large occurrence of off-target effects, which result from the inherent
characteristics of these approaches. Various techniques have been attempted and
rened to minimize the unintended impacts. Furthermore, because of the heterogeneous nature of the mutations produced by these approaches, it is necessary to separate and identify these changes. Furthermore, these techniques induce bi-allelic
mutation at the desired location. It would be impossible to generate mutant mice
using these methods if the mutations had a detrimental impact on embryonic development or neonatal survival. Despite signicant technological advancements and
several benets in generating GEMMs using these approaches, the applicability of
GEMMs created by these methods is limited to specic circumstances due to the
various difculties mentioned earlier.
10.3 Production ofTransgenic Animals
In the scientic literature, three distinct categories of experimental animal models
are discussed. These may be categorized as induced, spontaneous, and transgenic
models. Induced models are created by laboratory techniques such as administering
medicine or chemicals, providing specialized diets, or performing surgical operations. Spontaneous models arise as a result of spontaneous mutations, whereas
transgenic models belong to the third category (Bagle etal. 2012). Transgenic refers
to the procedure of introducing complementary deoxyribonucleic acid (cDNA)
obtained from specic messenger ribonucleic acid (mRNA) into cells (Welfare
NIoHOoLA, Association AREN 2002). Animals that have had their genes altered
by the insertion of recombinant DNA are called transgenic (Colman 1996).
The development of transgenic animals typically follows a similar sequence,
regardless of the species (Houdebine 2009; Mohan etal. 2017):
1. Determination and integration of the exogenous gene and associated promoter
sequences.
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2. Introducing DNA straight within the pronucleus of a single fertilized egg using
several techniques.
3. Transplantation of these genetically modied cells within surrogate mothers.
4. Demonstrating the successful development of the embryo till birth, conrming
the stable and inheritable integration of foreign DNA into the DNA of at least a
portion of the offspring at birth.
5. Establishing the gene’s efcient regulation in order to effectively operate in its
new surroundings.
Microinjections or transposon techniques may be used to introduce the foreign
DNA into the embryo’s cytoplasm or pronucleus. Sperm cells, cloning, lentivirus,
and pluripotent cells are other ways in which DNA may be transferred. The latter
three methods allow the incorporation of genes either randomly or by targeted integration using homologous recombination or gene substitution, leading to genetic
alteration (Houdebine 2009; Ghanghas etal. 2021). Targeted mutation refers to the
intentional modication of a specic gene, achieved by either eliminating or deactivating it (knocked out) or sometimes by rendering it functional (knocked in)
(Welfare NIoHOoLA, Association AREN 2002; Niemann and Kues 2007). A
multiple- transgenic organism is an organism that has been genetically transformed
with more than one transgene (Niemann and Kues 2007). These approaches do not
lead to the establishment of new species, but instead provide methods for producing
novel varieties of animals that contain distinct genetic information (Jube and
Borthakur 2006).
S. Banerjee et al.
10.4 Disease-Specic Transgenic Animal Models
Animal models are live, nonhuman organisms utilized for scientic study and
exploration of human ailments, with the aim of enhancing our comprehension of
sickness without subjecting a human being to potential injury throughout the
entirety of developing and discovering drugs. Fetal eggs are fertilized with human
DNA and then inserted into the oviducts of faux-pregnant females to develop into
transgenic animal models (Sulabh and Kumar 2018). Various illnesses have diverse
models of transgenic animals.
10.4.1 Angiogenesis
The use of mouse models allows for the study of vascular development in relation
to arterial stenosis, angiogenesis, thrombosis, atherosclerosis, thrombolysis, and
bleeding. This involves the assessment of various methodologies (Snaith and Törnell
2002). Currently, the suppression of the formation of new blood vessels represents
a signicant potential for the development of novel cancer treatments. Angiogenesis
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transgenic animal models are used to identify inhibitors that target particular pathways of angiogenesis (Snaith and Törnell 2002).
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10.4.2 Human Immunodeciency Virus (HIV)/Acquired
Immunodeciency Syndrome (AIDS)
The transgenic mouse model for testing some pathogenic manifestations is highly
useful in drug discovery research. The rst transgenic model was developed with
the express purpose of investigating HIV, where the Tg26 HIVAN Mouse Model
was released to the scientic community in 1991. Newer variants have been released
since then. Rosenstiel et al. provide a comprehensive overview of 32 transgenic
murine HIVAN models that have been established (Rosenstiel etal. 2009). These
transgenic animals have the ability to produce HIV-1 proteins and exhibit symptoms
and immunological decits that are comparable to the signs of AIDS in people.
There are two further versions available: the AIDS Mouse and the Smart Mouse
(Bagle etal. 2012).
10.4.3 Cardiovascular Disease
Given the wide range of models available, nowadays, the best strategy is to utilize
the transgenic models to investigate cardiovascular diseases by manipulating the
expression of genes involved in cardiovascular regulation, such as endothelin,
angiotensin, calcium binding-signaling, natriuretic peptides, catecholamines, nitric
oxide synthesis, and sodium channel transporters. These models allow for the study
of both gain and loss of function in these genes (Bader etal. 2000). Transgenic
models of heart failure and hypertrophy have been established, including gene overexpression of Calmodulin, gene mutation of alpha cardiac myosin heavy chain, and
knockout gene model of transforming growth factor (Hasenfuss 1998). A mutation
in the ApoE gene, which plays a crucial role in the absorption of chylomicrons and
very low-density lipoprotein particles, leads to the development of atherosclerotic
lesions that are histologically identical to those seen in humans (Snaith and
Törnell 2002).
Cancer
The oncomouse was the rst transgenic mammal to get a patent. For any cancer
drug, although they are active during preclinical studies, the nuances of testing these
drugs in the whole animal setting are equally important. The germ cells and somatic
cells of the animal include a human oncogene sequence that was introduced at the
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S. Banerjee et al.
early embryonic stage. This assures the presence of the oncogene in every cell of the
organism (Bagle etal. 2012). Tumor development and metastasis may be facilitated
by E-cadherin and other adhesion molecules by using different transgenic deletion
mice (Welfare NIoHOoLA, Association AREN 2002).
Alzheimer’s Disease
Prior to the use of transgenic technology, there were no animal models available for
the condition. Alzheimer’s disease is also regarded as the ideal disease for modeling
in transgenic animals. Vaccinating transgenic mice with Amyloid precursor protein
A42 demonstrated the promise of immunization as a treatment strategy for
Alzheimer’s disease. In order to better understand Alzheimer’s disease, several animal models have been developed. These include Alzheimer’s mice; TAU transgenic
mice such as 7TauTg and ALZ7 mice; amyloid pathology animals such as Tg2576
and PDAPP mice; and presenilin transgenic mice such as ApoE knockout (Spires
and Hyman 2005; Götz etal. 2004; Schenk 2002).
Diabetes Mellitus
In the transgenic mice, during the study, a variety of mechanisms ranging from biochemistry to histopathology and different models are used to represent the diversity
seen in human diabetic patients. It is necessary to construct transgenic models in
order to examine the genes involved in peripheral insulin action. Various models
have been created to study insulin secretion in type 2 diabetes, including glucokinase, islet amyloid polypeptide, and hepatic glucose synthesis (Srinivasan and
Ramarao 2007). A transgenic mouse model has been designed to exhibit InsulinDependent Diabetes Mellitus by introducing a viral gene during the animal egg
stage (Etuk 2010). Additional models, such as the beta receptor knockout mice and
the uncoupling protein (UCP1) knockout mouse, exist for evaluating antidiabetic
drugs. Furthermore, there are acute and chronic models available for this purpose
(Srinivasan and Ramarao 2007; Henson and O'Brien 2006; Kumar et al. 2012;
Eddouks etal. 2012).
Transgenic animal models are employed to evaluate mutagenicity and carcinogenicity, as well as to investigate metabolic enzymes and receptors (Boverhof etal.
2011). The World Health Organization has authorized transgenic animal models for
mutagenicity experiments, such as the LACI transgenic model (using the Big Blue®
construct) and the LACZ transgenic model (using the Muta™ Mouse construct). A
diverse array of transgenic animals has been created using several approaches in the
eld of experimental immunotherapy for cancer. Each of these tactics is designed to
stimulate distinct components of the immune system. Several of these models consist of transgenic rodents that express tumor-associated antigens, such as MUC1
transgenic mice, as well as transgenic mice with oncogenes to investigate immunotherapeutic approaches. Additionally, there are transgenic mice that express immune
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effector cell molecules, such as Fc-receptor transgenic mice (McLaughlin et al.
2001). The preclinical transgenic model investigates the bioactive products of
Matrix Metalloproteinase (MMP) inhibitors and their potential impact on cellular
function (Pavlaki and Zucker 2003). Animal models are also created for skeletal
muscle disease, Huntington’s disease, and other disorders (Ramaswamy etal. 2007;
Horton 2003). Medical research requires disease models to identify specic targets
for medication development. Manipulating the genetic composition of these animals by adding or deleting genes grants them novel characteristics that become
valuable in gaining a deeper comprehension of diseases or developing curative solutions. Conducting the rst testing in people is both unethical and unsafe; hence,
transgenic animals are used instead. Animal research is essential for the rst evaluation of novel vaccinations and treatments, making disease models invaluable
(Tiwari etal. 2012).
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10.5 Transgenic Animal Products
The primary focus of studying transgenic organisms is to explore their scientic
uses and evaluate their potential for commercial pharmaceutical production. Below
are a few examples of transgenic animals now undergoing research and the goods
they are expected to produce.
Cow: Factors-VIII and -IX, recombinant antithrombin III (rATIII), protein C,
human milk protein, and recombinant human serum albumin (HSA) (Lillico
etal. 2005).
Chicken and Egg: Interferons, cytokines, vaccines, insulin, HSA, and monoclonal antibodies (MAbs) (Lillico etal. 2005).
Goat: MAbs, tPA (tissue Plasminogen Activator), Ig fusion proteins, and ATryn
(recombinant human antithrombin III) are examples of transgenic recombinant proteins. ATryn, in particular, has the distinction of being the rst transgenic recombinant protein from a transgenic animal to get approval from the US Food and Drug
Administration (FDA) in January 2009 (Lillico etal. 2005; Ormandy etal. 2011).
Sheep: Sheep milk contains brinogen, which is a key component along with
thrombin and Factor XIII.It also contains human Factor-VII, -IX, α1-antitrypsin,
and activated protein C (Lillico etal. 2005; Hunter etal. 2005).
Rabbit: Recombinant forms of human C1 inhibitor, human α-antitrypsin, human
erythropoietin, tPA, α-glucosidase, human interleukin 2, and human growth hormone (Lillico etal. 2005).
Mice: Production of many proteins for the creation of a malaria vaccine. These
proteins include ATIII, MAbs, β-interferon, Factor X, myelin basic protein, HSA,
cystic brosis transmembrane regulator, brinogen, tPA, prolactin, and antineoplastic urine protein (Lillico etal. 2005).
Different Other Species: Some of the most often used model species for
studying promoter elements and gene transfer strategies include frogs, nematodes, and marine invertebrates (Lillico etal. 2005). At present, the majority of
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pharmaceutical firms verify a significant number of targets to different degrees
and advance them to the high-throughput screening stage. The medications that
target these specific sites are then used in clinical trials, which often have a
high incidence of attrition, resulting in significant costs. Increasing the use of
transgenic models is thought to decrease the volume of work needed to achieve
success, resulting in a substantial reduction in expenses (Jube and
Borthakur 2006).
S. Banerjee et al.
10.6 Applications ofDrugs Derived fromTransgenic Animals
andOther Sources
The use of proteins as medications started in the 1920s when insulin derived from
pig pancreas was rst employed. During the early 1980s, scientists produced
human insulin using recombinant bacteria, which is today the primary form of
insulin used by the majority of diabetes patients. The success was restricted because
bacteria lack the ability to produce intricate proteins like monoclonal antibodies or
coagulation blood factors, which need posttranslational changes to become functional or stable inside a living organism. These objectives can only be completely
accomplished in mammalian cells that can be cultivated in fermenters or used in
live animals. Currently, two methods are being deployed for the production of
recombinant proteins in transgenic animals. The rst option is the milk produced
by transgenic animals, while the second option is the chicken egg white method.
Transgenic animals are being used to generate a diverse range of recombinant proteins, including vaccines, cytokines, blood factors, antibodies, hormones, brinogen, milk proteins, growth factors, enzymes, collagen, and more (Houdebine 2009;
Hunter etal. 2005).
The mammary gland is the optimal location for production due to its ability to
create large amounts of protein and the availability of well-established techniques for extracting and purifying these proteins. In order to study the genes
involved in peripheral insulin action, transgenic models are developed. The
α-glucosidase enzyme derived from transgenic rabbits’ milk has effectively
treated Pompe’s illness (Niemann and Kues 2007; Hunter et al. 2005). Other
potential systems for analysis include the silk gland, seminal plasma, blood,
urine, and insect larvae hemolymph. Blood often has limited capacity to retain
elevated amounts of recombinant proteins, which are inherently unstable.
Additionally, the presence of biologically active proteins in the blood might
potentially impact the health of the animals. Milk is now the most advanced
technique for producing recombinant proteins from transgenic species, thereby
circumventing these issues. Current investigations conrm that egg whites may
serve as a viable source of foreign proteins, including recombinant vaccines
(Houdebine 2009; Hunter etal. 2005).
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