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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5918_Библиотеки_им_академика_М_И_Перельмана
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Part II
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Sec B: Current Trends of Biotechnology in
Drug Development
Chapter 9
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Transgenesis: IntheDrug Discovery
Process, Including Target Identication
andTarget Validation
AniruddhaMukherjee, SusovanDas, NurulHassanMondal,
AvijitChoudhury, andSugatoBanerjee
Abstract Transgenic targeting is the most dependable method for mouse trans-
genesis because it produces predictable transgene expression patterns. This
method is ideal for large-scale knock-in target assessments. Furthermore, transgenic targeting is the preferred method for getting around the restrictions since it
is a powerful cellular delivery strategy. The chapter introduces the concept of
target validation and underscores its signicance in evaluating a target’s potential
for therapeutic benets. The focus is on improving the drug development process
through early target validation. Target validation, a critical initial step taking few
months, involves proving that altering the target in animals and cell cultures signicantly improves the disease phenotype. Target validation in transgenic animals
is explored in detail, emphasizing the necessity of clearly dening a model’s
objectives for appropriate creation, evaluation, and application. The difculties of
accurately simulating human genetic abnormalities in transgenic animals are discussed despite the possibilities offered by genome editing. The importance of
A. Mukherjee (*)
School of Pharmacy, The Neotia University, Sarisa, West Bengal, India
S. Das
Acharya & BM Reddy College of Pharmacy, Bengaluru, India
N. H. Mondal
Dr. B.C. Roy College of Pharmacy and AHS, Dr Meghnad Saha Sarani,
Durgapur, West Bengal, India
A. Choudhury
Gupta College of Technological Sciences, Asansol, West Bengal, India
S. Banerjee
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education
and Research, Kolkata, India
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_9
159© The Author(s), under exclusive license to Springer Nature Singapore Pte
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validity in assessing animal models is reiterated, advocating continuous debate
and improvement. The translational method is emphasized for differentiating
between target validations in humans and qualifying in animals. Face and construct validity in animal models are explained, with the latter deemed more crucial. The challenges of simulating human genetic disorders in transgenic animals
are acknowledged, with the recommendation to use diverse models to improve
predictive validity.
Keywords Transgenic target identication · Transgenic target validation · Face
validity and construct validity
A. Mukherjee et al.
9.1 Introduction
Transgenesis is a process of experimentation in which foreign genes are incorporated into the genome of an organism, resulting in the creation of pathological conditions for the observation of new drug therapeutic potential. However, transgenesis
is also used to disrupt genes (knockout). The process of creating a new drug from
scratch can take more than 10–15years with uncertainty and exceed one billion dollars (based on the therapeutic application area) (Hughes etal. 2011). To increase the
success rate, it is very crucial to use the whole organism early on in the process of
nding new drugs. Transgenesis is the most prominent choice for the early stage of
the drug discovery process because most of the drugs fail to pass phase II clinical
trials because they do not meet the standards of ADME due to the multiple interactions of the invivo study (Singh and Seed 2021).
The roots of pharmacology and medicine trace back to ancient civilizations,
where the use of medicinal plants and natural remedies formed the foundation of
early healthcare practices (Wadud etal. 2007). Later on, the evaluation of synthetic
chemistry will strand the new drug development process. This conventional process
of drug discovery has many limitations, including a higher rate of failure during
testing, limiting target specicity, and, most importantly, taking many years to
develop a new drug. To overcome the limitation, transgenesis is an emerging eld.
There are various methods available for creating transgenic animals, including
microinjection, viral vectors, embryonic stem cell-mediated transgenesis, and
CRISPR/Cas9 technology (Costantini 2001).
Target validation and identication are just two of the many phases of the investigation procedure that transgenic technology can help in the procedure for nding
new drugs. In the case of target identication in transgenic animals carrying big
DNA, fragments can be utilized to focus on genetic areas (Snaith 2002). In the case
of target validation, transgenesis allowed the creation of genetically modied
organisms to mimic human diseases, enabling a deeper comprehension of the
underlying mechanics and potential treatments. Additionally, transgenic models
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have revolutionized drug discovery by serving as reliable platforms for testing new
treatments before they are applied to human patients for a better understanding of
disease mechanisms (Lee 2014).
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9.2 History
9.2.1 Ancient Roots ofMedicinal Plant Knowledge
The history of medicinal plants and their uses dates back to ancient times, and
many cultures have relied on herbs and natural remedies for their medicinal properties. In Islamic tradition, there is knowledge of the properties of various herbs
and their healing abilities were passed down by Allah through Adam (Alaihissalam)
as the rst messenger (The Qur’an 2023). Throughout history, humans have utilized plants, minerals, and animal-derived substances for medicinal purposes.
Traditional herbal medicine practices laid the foundation for early drug discovery.
Alternatively, during early civilizations, clay tablets from Sumeria listed hundreds
of medicinal plants, while the Ebers Papyrus from Egypt describes over 850 plantbased medicines. During the eighteenth century, the establishment of the periodic
table by Dmitri Mendeleev and Avogadro’s atomic hypothesis laid the foundation
for understanding the building blocks of molecules and their properties. Later, one
isolation of morphine from opium extracts by Sertünergame changer for development of new drug compound (Drews 1979). The isolation of individual bioactive
ingredients from plant materials, even when not meeting modern pharmaceutical
standards, dramatically improved Byronic period medicine. In the Byronic period,
medicine took a giant leap forward with the isolation of individual bioactive ingredients from plant materials despite limitations in purity (Drews 1979; Beale and
Block 2011).
9.2.2 Twentieth-Century Chemical Knowledge
The twentieth century saw signicant advancements in chemical knowledge, including the concept of aromaticity in benzene, which signicantly inuenced Paul
Ehrlich’s theory of “chemoreceptors.” J.N. Langley presented the functional theory
of receptive substances in 1905, which enables receptors to receive signals and have
pharmacological or biological effects. This study spurred more investigation into
medicinal chemistry, which helped establish the concept of ligand-receptor interaction (Drews 1979). Ehrlich’s research opened the door for the rst sensible synthetic
medications, including arsphenamine, in 1908. Furthermore, Ehrlich’s research
group established the framework for credible biological screening and assessment
methods (Beale and Block 2011).
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A. Mukherjee et al.
9.2.3 Antibiotics andDrug Discoveries
Before NMR and computational chemistry, chemists had limited tools, making
institutional support crucial for drug discovery. The 1928 discovery of penicillin, a
blend of interconnected beta-lactams, by Alexander Fleming revealed its potent
antibiotic properties. By 1942, mass production began, signicantly affecting World
War II sepsis management. Allied medical personnel carried penicillin during the
operation, and it remains an indispensable tool in combating infections caused by
Gram-positive microorganisms today (Newman etal. 2000).
A novel compound called cephalosporin was rst described by Brotzu in 1948
and was used to treat infections resistant to penicillium. By that time, other businesses had expanded their microbiological capabilities, including Merck, Sandoz,
and Taked, in an effort to discover alternative medications with distinct pharmacological and chemotherapeutic qualities (Drews 1979).
Starting in 1950, the landscape of medicinal chemistry underwent a pivotal transformation, delineating two signicant epochs: Initially, spanning from 1950 to
1980, medicinal chemistry thrived on invivo testing, while the subsequent era,
commencing in 1980 and persisting to the present day, heralded the rise of pioneering design methodologies and advanced screening technologies (Drews 1979;
Lombardino and Lowe 2004).
During the late 1960s and early 1970s, Beecham stumbled upon clavulanates,
while Pzer crafted sulbactams, molecules akin to penicillin in how they acted
within the body. Beecham’s discovery mirrored penicillin’s traits, while Pzer
synthesized sulbactam by altering the sulfur in thiazoles to sulfone, creating a
semi- synthetic compound (Newman etal. 2000). During this phase, a key focus
involved assessing how drugs interacted within the human body. Despite discovering promising compounds that exhibited activity within animal models, challenges persisted in understanding how these substances were absorbed, distributed,
metabolized, excreted, and interacted within the clinical setting (Lombardino and
Lowe 2004).
In 1974, Annie Chang and Stanley Cohen achieved a groundbreaking feat by
introducing Staphylococcus aureus genes into Escherichia coli, marking the birth of
the inaugural transgenic organism (Chang and Cohen 1974). Gene transfer occurred
in yeast cells for the rst time among eukaryotic organisms back in 1978 (Hinnen
etal. 1978). In 1979, the transformation of mouse cells marked a signicant breakthrough, swiftly followed by the transformation of mouse embryos in 1980. Initially,
scientists primarily employed microinjection, directly infusing DNA into cells, for
these groundbreaking transmutations. However, as research progressed, diverse
techniques emerged: integrating transgenes into retroviruses for cell infection,
leveraging electro-infusion by utilizing electric currents to introduce foreign DNA
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across cell walls, employing biolistic—a technique that involves injecting DNA
“bullets” into cells, or even directly putting DNA into freshly fertilized eggs. These
varied methods expanded the repertoire of transformation techniques, enriching the
eld of genetic manipulation (Evans 2011).
Transgenes consist of three key components: a polyadenylation signal for
mRNA stability and protein expression, a promoter/enhancer, and the sequence
encoding the intended expression product (such as a reporter protein or regulatory RNA). While detailing the complete history of recombinant DNA technology is impractical, signicant milestones include the development of enzymes
enabling DNA fragment manipulation and recombination, DNA fragment cloning in bacteria leading to protein expression, chemical synthesis of DNA such as
oligonucleotides, and the polymerase chain reaction’s (PCR) invention (Xu
etal. 2019).
Once scientists gained access to recombinant DNA technologies, the natural progression was to explore introducing this modied incorporating DNA into model
systems such as organisms or cells in culture like mice. Early experiments demonstrated the feasibility technique of transfecting grown cells with externally cloned
DNA to produce proteins. These studies utilized drug selection methods to pinpoint
transformed cell clones containing the genetic material and expressing thymidine
kinase of the herpes simplex virus (Durmaz etal. 2015).
The technique of transforming cultured cells with DNA using the process of
precipitating DNA using calcium phosphate, detailed in these studies, proved effective for cultured cells but was not applicable to zygotes, the early stage of a fertilized egg (Kwon and Firestein 2013).
The creation of transgenic mice via DNA microinjection into mouse zygote
pronuclei necessitated the convergence of multiple research disciplines. It
involved aspects of mouse reproductive physiology (such as superovulation,
embryo culture, and surgical transfer to pseudopregnant females), specialized
micromanipulation equipment, and molecular genetics. Initially, microinjection
techniques were employed to evaluate the developmental potential of blastomeres
from two-cell and four-cell embryos by lysing some blastomeres and observing
the remaining ones after implantation into pseudopregnant recipients (DeMayo
etal. 2012).
The rst microinjection experiment on mouse zygotes assessed the survival
and development of eggs given a gamma globulin injection post-transfer.
Microinjection found various applications, including studies on oocyte biology,
translation of foreign mRNA species, and preimplantation developmental biology.
As DNA cloning became a standardized method in molecular biology, all the necessary components for producing transgenic mice were established. Subsequently,
following the initial successful creation of transgenic mice, the eld rapidly
expanded (Saunders 2020).
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A. Mukherjee et al.
9.2.4 Advancements inGenetic Engineering
The advancement in genetic engineering technology has seen a transition from
ZFNs and TALENs to the now-prevalent CRISPR/Cas9 technology because of its
exceptional adaptability and user-friendliness. CRISPR/Cas9 technology has revolutionized genome targeting, particularly in the genetic modication of mice and
rats. Its delivery via various forms, such as Cas9 ribonuclease protein complexes,
has shown advantages, minimizing unwanted genome integration and reducing
mosaicism in animal founders (Gupta and Musunuru 2014).
CRISPR/Cas9’s efciency in inducing gene knockouts or precise modications
such as point mutations, epitope tags, and conditional genes has signicantly
streamlined the generation of genetically modied animals. Techniques utilizing
linear double-stranded DNA templates or lengthy single-stranded DNA molecules
have notably improved efciency, especially in producing oxed alleles compared
to earlier methods (Arora and Narula 2017).
Controlling off-target mutations in the genome is crucial when employing
CRISPR/Cas9. Careful guide RNA sequence selection and utilizing specialized
Cas9 enzymes, such as enhanced specicity Cas9 or high-delity Cas9, have been
pivotal in minimizing off-target effects. Analysis methods beyond the founder generation require meticulous attention to avoid misinterpretations due to natural
genetic drift during development (Guo etal. 2023).
Despite its profound impact, CRISPR/Cas9 technology is not immune to potential replacements in the future. Alternative tools such as Cpf1 enzyme have shown
promise but are less efcient than Cas9. Researchers continuously explore and optimize CRISPR/Cas9 while anticipating the possibility of even more precise and
active genome editing tools down the line (Wang etal. 2022).
The overarching impact of CRISPR/Cas9 on creating genetically modied models for research is undeniable, yet the eld remains open to future innovations that
may further enhance precision and efciency in genome editing (Mengstie and
Wondimu 2021). Some approved and viably produced biopharmaceuticals obtained
from genetically modied animals are included in Table9.1.
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165
(continued)
Wang etal. (1995)
Biotherapeutics
Inactive or terminated Bayer-PPL Brantly etal. (1988)
(2008)
Merrimack- GTC Debruyne and Delanghe
Phase II
Abgenix- Amgen Yang etal. (2001)
Murphy and Atala
Braunwald (1988)
(2017)
Prior to clinical Nexia Darvesh etal. (2003)
(1995)
(2014)
Novartis Mayo Clinic
Alpha-1 antitrypsin A genetic lack of a protein that shields the
Anti-C5 monoclonal antibody Kidney inammation, rheumatoid arthritis Prior to clinical Alexion-GTC
Item Therapeutic or other use Stage of development Establishment Reference
Table 9.1 Biopharmaceuticals obtained from genetically modied animals
lungs’ elastic components results in
emphysema, a hereditary illness that
seriously affects the body’s digestive
system, lungs, and other organs
As a result of antibodies blocking
nerve-muscle transmission, the skeletal
Alpha- fetoprotein (AFP) tumor
marker
Cancer Phase II
muscles of people with multiple sclerosis,
a chronic autoimmune illness, get weaker
Rheumatoid arthritis Inactive or terminated Abgenix-GTC Huang etal. (2002)
Anti-EGF receptor monoclonal
antibody
ABX-IL8 recombinant human
combat biological threats, and prepare for,
respond to, and recover from bioincidents
Butyrylcholinesterase Those measures intended to mitigate risks,
Antithrombin III There are several plaque fragments or clots Phase III GTC Bick (1982)
monoclonal antibody
Prolactin Bolstering of immunity I-T GTC Freeman etal. (2000)
Rotavirus virus-like particles Vaccine development Preclinical Bioprotein Changotra and Vij
Tissues or organs Designed with xenotransplantation in mind Preclinical Alexion BresaGen
Activator of tissue plasminogen Breakdown of blood clots Inactive or terminated Genzyme Loscalzo and
Lactoferrin Anti-inammatory Phase I Pharming Levay and Viljoen
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A. Mukherjee et al.
(1998)
ImmunoGen- GTC Wang etal. (2005)
(1994)
(1991)
Nozik- Grayck etal.
(2005)
(1984)
Various indications Preclinical Hematech Avigenics Vaswani and Hamilton
Polyclonal antibodies that are
humanized
1 on the surface of merozoites Malarial vaccine Preclinical GTC Lin etal. (2014)
Item Therapeutic or other use Stage of development Establishment Reference
Table 9.1 (continued)
huN901-DM1, ImmunoGen Lung cancer with little cells Preclinical
Glutamic acid decarboxylase Type 1 diabetes Inactive or terminated GTC-Biosyn Erlander and Tobin
Glucagon-like peptide-1 Type 2 diabetes Inactive or terminated PPL Holst (2007)
Ischemic reperfusion injury Inactive or terminated Pharming
The superoxide dismutase
Human growth hormone Turner’s syndrome Inactive or terminated GTC Strobl and Thomas
extracellular
Glucosidase Glycogen storage disease Inactive or terminated Pharming Lebovitz (1997)
G-CSF Leukopenia Preclinical Avigenics Mehta etal. (2015)
Natalizumab Multiple sclerosis and Crohn’s disease Preclinical Elan-GTC Brooks and Walker
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9.3 Transgenesis: Techniques
In the intricate tapestry of genetic exploration, transgenic animals stand as invaluable allies, unraveling the secrets encoded within our DNA.By introducing exogenous genetic material into fertilized zygotes and guiding their journey through
gestation, scientists have forged a powerful tool in the pursuit of understanding gene
function and mimicking human diseases (Houdebine 2003). Transgenic models
serve as dynamic canvases painted with specic genetic mutations that echo the
intricacies of human diseases. They not only offer profound insights into molecular
and physiological mechanisms but also validate potential drug targets, probe drug
pharmacokinetics, and scrutinize the safety and immunogenicity of promising candidates, expediting the labyrinthine journey of drug development (Mayran and
Bolt 2022).
These genetically crafted cohorts are not mere witnesses; they are active participants in the relentless pursuit of knowledge. Beyond facilitating the understanding
of specic genes and paving the way for personalized medicine, transgenic models
play a pivotal role in scrutinizing the labyrinthine landscape of cancer development,
progression, and responses to treatment (Wijmenga and Zhernakova 2018).
As the shadows of neurodegenerative diseases loom large, transgenic models of
ailments such as Alzheimer’s and Parkinson’s emerge as beacons of hope. They not
only decipher the intricate dance of disease mechanisms but also serve as testing
grounds for potential therapeutic interventions (Rani etal. 2023).
In the ever-evolving eld of gene therapies, transgenesis takes center stage, providing a meticulous testing ground for the efcacy and safety of these groundbreaking approaches before they venture into the uncharted territory of clinical trials
(Dunn etal. 2005).
9.4 Types ofTransgenic Techniques
From the pioneering work of Herbert Boyer and Stanley Cohen in the 1970s, since
the development of recombinant DNA technology, and during the past few decades,
numerous techniques for creating transgenic animals have been created (Wright
1986). In the realm of drug discovery, the convergence of genomics, proteomics,
and advanced reproductive biotechnologies has paved the way for groundbreaking
transgenic applications in domestic animals. The methodologies employed in creating transgenic animals are meticulously tailored based on the specic intended purpose of the animal in the drug development process. There are three types of
transgenic techniques for the transfer of foreign gene: gene transfer-based transgenesis, vector-based transgenesis, and targeted integration of foreign genes (Spreaco
etal. 2020).
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