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. • PROTAC: How to force therapeutically untargetable proteins into targeted degradation
tin-mediated amyloidosis. Worldwide, approximately
50,000 people suffer from this rare and extremely devastating disease. Patisiran successfully delivers siRNA to
liver cells using lipid nanoparticles as adelivery vehicle.
12.8 PROTAC: How to force
therapeutically untargetable proteins
into targeted degradation
Traditional small molecule drug discovery for intracellular targets has focused on developing high-afnity ligands that target either an active, orthosteric, or allosteric
site on a target protein to interfere with its physiological
function. While this approach has been highly effective, it
has left potential drug targets undruggable because their
active sites are either too broad, formed as shallow pockets, or have at, unstructured surfaces that provide very
few sites with signicant ability to bind small molecules.
Instead, the PROTAC (Proteolysis-Targeting Chimeras) approach involves the targeted degradation of a given
protein to down-regulate or completely shut off its biological function in the organism. It induces the selective
proteolytic degradation of pathogenic and, therefore, unwanted proteins into amino acids by the cell’s own proteolytic degradation machinery (proteasome, Sect.23.8). To
be degraded by the protein shredder, aprotein must rst
be labeled. This is done by attaching ubiquitin markers to
the protein (the so-called “kiss of death”). This requires
several of these ubiquitin peptides, which are added one at
atime. Abifunctional molecule (“chimera”) is required to
capture the protein to be degraded and to mark it for degradation. In PROTAC molecules, this is achieved by means
of a chemical linker (e.g., a polyethylene glycol chain)
connecting the two functional ends. On one side, it carries
adecoy that binds the target protein to be degraded. On
the other side, it carries another decoy that recruits an E3
ubiquitin ligase to do the labeling. The function of the E3 ligase, thus, determines which protein becomes the substrate
for proteolytic degradation in the proteasome. Since the
PROTAC molecule brings the protein to be degraded into
spatial contact with the E3 ligase and, thus, with the “molecular machinery” for ubiquitin labeling (. Fig.12.4), the
protein to be degraded becomes asubstrate for aligase.
Once the captured target protein has been labeled and targeted for degradation, the PROTAC molecule is released.
Like acatalyst, it can then initiate the next cycle of degradation. Unlike aclassical inhibitor, which must inactivate
the target protein in stoichiometric amounts, PROTAC
molecules are effective at lower dose levels. It appears that
proteolytic degradation takes some time, but this does not
necessarily reduce the success of the therapy.
A moderate afnity of the decoy to capture the target
protein to be degraded is usually sufcient, making the
design of such a decoy ligand easier. On the other hand,
the ligand moiety that recruits the E3 ligase should be a
potent binder with a slow off-rate. PROTAC molecules
that attach even covalently to an E3 ligase may be superior because they reduce the three-body assembly kinetics
to a two-body case, thereby increasing catalytic efciency.
The attractiveness of this concept is that once adecoy
for the protein to be degraded is found, the PROTAC
. Fig. 12.4 Crystal structure of acomplex
of aVHL-E3 ligase (light blue, von Hippel–
Lindau tumor suppressor with ubiquitin
ligase E3 activity and two further domains,
elonginB andC) and atarget protein
BRD4 (magenta, epigenetic target protein,
abromodomain that can read acylations on
histone proteins, see Sect.12.14) to be ubiquinylated. The PROTAC molecule can be
seen in the center and links the two proteins
via its decoy groups (orange/blue). Chemical-
ly, the two decoys are linked via apolyethylene glycol (PEG) inker (purple). The two
decoys each t highly specically into the
pockets of the ligase and the bromodomain,
respectively. (7 https://sn.pub/8AwGNS)
Chapter • Gene Technology in Drug Research
12
system can force any protein to be degraded in this way.
This includes proteins previously thought to be therapeutically undruggable. Any small molecule ligand that
binds specically to the surface of the target protein can
serve as adecoy, without the need to possess a functional
role on the target protein.
It also needs an E3 ligase to capture the PROTAC
molecule using its second decoy. Although there are over
600 E3 ligases in our genome, only afew can be captured
using asmall synthetic ligand as adecoy. One of these
is thalidomide (5.26, Sect.5.5). It binds to the cereblon
protein complex (CRBN), which has several functions in
the body. During embryonic development, for example,
it forms the substrate-binding subunit of aubiquitin E3
ligase that inuences limb growth by regulating transcription. This has been implicated in the teratogenic effects
observed with thalidomide. However, there are several
other examples of such E3 ligands that can be used to
synthesize adecoy in PROTAC molecules. . Fig.12.4
shows the structure of aternary complex of another E3
ligase (VHL-E3 ligase), aPROTAC molecule with two
specic decoy groups, and the epigenetic target protein
BRD4.
Most PROTAC molecules described so far in the
literature focus on epigenetic and oncogenic targets
(Sect.12.14), nuclear hormone receptors (Sect.28.1), or
kinases (Sect.26.2). In 2019, the rst PROTAC molecule
entered clinical testing, in 2020, these trials provided the
rst clinical proof-of-concept against the estrogen (ER)
and antrogen receptor (AR) as well-established cancer targets. PROTAC molecules have alarge molecular weight
and their physicochemical properties are outside the typical
range of small molecule drugs (see Rule of Five, Sect.19.7),
which may pose challenges for drug delivery into the cells.
Nevertheless, examples with sufcient oral bioavailability
have been reported and rst candidates have reached phase
III clinical trials. Recent research extends to so-called
bioPROTAC molecules, which use peptide-like decoys to
lure target proteins for selective degradation. There is no
doubt that the PROTAC concept has great potential and
will hopefully prove itself in therapeutic applications.
12.9 Proteomics and Metabolomics
The approaches described in Sects.12.5 and12.7 aim to silence adisease-causing gene or agene that plays an essential
role in adisease. But how do we know whether aparticular
gene or gene product is involved in adisease process at all?
Decisive indicators to answer this question can be extracted
from the protein composition in the cell. This composition
changes dynamically. It is called the proteome and reects
the totality of all proteins in acell, or even in the entire
organism, at agiven point in time under well-dened conditions. Focusing on the protein pattern of acell from aparticular organ, important variables are the metabolic state,
the developmental stage of the organism, the current stage
in the cell cycle, and the surrounding temperature. Disease
processes and drug therapy also alter this pattern. In the
genome, all theoretically expressed proteins are encoded as
static genetic information. In contrast, the proteome reects
the protein composition at aparticular point in time. The
difference between abuttery’s caterpillar and adult stages
is astriking example of the difference between the genome
and the proteome. The genome is the same for both, but the
proteome is signicantly altered, resulting in acompletely
different phenotype (caterpillar/buttery).
The proteome can be used to compare the state of
healthy cells, diseased cells, and cells under the inuence of
drug therapy. At rst glance, this seems to be an extremely
complex and almost impossible task. A cell contains
thousands of proteins, many of which are modied after
they are expressed. For example, the rst amino acids of
asequence are cleaved (Sect.25.9), phosphate groups are
transferred (Sect.26.3), sugar building blocks are added,
disulde bridges are coupled, prosthetic groups are added,
and ubiquitin or prenyl groups are added (Sect.26.11). In
addition, alternative RNA splicing occurs as amechanism
of gene regulation, further increasing the diversity of the
proteome from acomparatively small number of genes.
All this dramatically increases the diversity of the protein
composition, probably by afactor of 5–10 compared to
the genome composition. Nevertheless, asophisticated analytical method has been developed that makes it possible
to analyze the proteome of acell at agiven point in time.
First, the proteins of acell must be denatured in such
away that all modifying processes are abruptly stopped so
that conclusions can be drawn about the cell contents. The
cell lysate is then subjected to separation. Proteins contain
many acidic and basic amino acids, so for each protein there
is awell-dened pH value at which protonation or deprotonation reaches astate where the protein appears to be
electrically neutral (isoelectric point). This pH is specic for
each protein and depends on the amino acid composition.
The protein mixture is added to asolid support (apolyacrylamide gel) such as is typically used in chromatography.
Avoltage is then applied. If the proteins carry acharge,
they will migrate across the solid support in the direction
of the oppositely charged pole. Thus, at some point during
their migration across the gel, which is designed to provide
acontinuous pH gradient from one end to the other, the
applied proteins will reach apoint where their entire exterior appears uncharged. When this position is reached on
the solid support, the proteins no longer migrate. The proteins are therefore separated according to their isoelectric
point, aprocess known as isoelectric focusing. All proteins
with the same isoelectric point migrate the same distance
and appear as amixture. The chromatography plate is then
rotated by 90° and the proteins are separated once again
but using adifferent principle. The proteins are thermally
denatured and their charges are masked with sodium dodecyl sulfate, ahighly charged anionic surfactant, so that
. • Proteomics and Metabolomics
. Fig. 12.5 2D-Gel electrophoresis for acellular proteome analysis.
Left Proteome of anormal cell. Center Proteome of apathologically
altered cell. Right Proteome of apathologically altered cell after treatment with adrug. Changes in protein concentration are indicated by
red circles. Above all, the proteins at positions3, 6, and7 are clearly
they are all virtually equally charged on the outside (SDS-
PAG E ). When an electric eld is applied, the denatured
proteins migrate again. The speed of migration depends on
the mass of the proteins. The direction of migration, which
is perpendicular to the rst isoelectric separation, results
in the initial proteome being broadly distributed and well
separated on the solid support.
Using this 2D electrophoresis, it is possible to separate
many thousands of proteins. The amount and sequential
composition of the separated proteins must be characterized. Many different staining and uorescence techniques
have been developed for quantitative analysis. They allow
quantitative determinations, especially in comparison to
the proteomes of analogous cells in adifferent state. Thus,
aquantitative comparison of the protein composition in
adiseased and ahealthy state is possible. It is also possible
to determine how the proteome changes under the inuence of adrug (. Fig.12.5). But how can one nd out
what is contained in each individual protein spot on a2D
gel? The answer is to extract the proteins from the plate
and digest them with trypsin. This protease (Sect.23.3)
cleaves the denatured proteins into small peptide fragments, which are then analyzed by mass spectrometry.
Sophisticated technologies combined with computer
analysis of precalculated protein fragmentation patterns
allow the reconstruction and sequence characterization
of proteins. Proteins in the proteome that are either up- or
down-regulated due to adisease process can be detected
in this way. However, whether the altered expression pattern causes or is aconsequence of the pathological state
remains to be determined by independent experiments.
As described above, the proteome of acell can be altered
by adrug. What are the interaction partners for agiven
drug? Will the induced effects always be the same if drugs
of the same class are used? In the research group of Giulio
Superti-Furga in Vienna, Austria, the properties of three
up-regulated in the diseased state. Afew of the pathological changes
are corrected by the drug therapy, but new changes in the proteome
(e.g.,2, 8, and10) might be induced by side effects. (Figure from Lottspeich, Angew. Chem. Intl. Ed. Engl., 38, 2476–2492 (1999) with the
kind permission of the author and publisher)
kinase inhibitors developed for the treatment of chronic
myeloid leukemia (Sect.26.4) were investigated in detail.
First, the drug had to be equipped with achemically inert
anchor group. It is certainly achallenging task to nd the
right position for an anchor on such acompound so that
the mode of action is not signicantly disturbed. Usually,
several positions along the molecular scaffold have to be
tried and tested. Finally, the drug is irreversibly covalently
coupled to achromatography column via the attached anchor group. The column is then loaded with the proteome
of acell lysate. Proteins that have afnity for the immobilized drug will adhere to the column. Finally, the binding
partners detected in the pull-down experiment have to be released from the column, separated, and characterized analogously to the technique described above. The composition
of all proteins with afnity to the tested drug is obtained. It
is difcult to make quantitative statements about the afnity of the binding partners, especially since the amount of
proteins and their composition in the lysate are highly variable. However, it is possible to construct aprole for each
compound according to its protein interaction partners.
This led to the surprising result that even drugs belonging
to the same or similar substance classes and developed for
the same therapeutic indication can exhibit signicantly dif-
ferent interaction proles in the cell. This is an impressive
observation, the evaluation and application of which will
require agreat deal of research. We will see in the next section that this can explain the different efcacy, therapeutic
deviations, and variable side effect proles in patients.
Proteomic techniques can also be used in clinical diagnostics. Without the exact resolution of the analyte,
signicant changes can be detected in the form of angerprint of the distribution of molecular masses. Tumor
diseases are revealed by changes in their protein composition. These can be detected at avery early stage, hopefully allowing curative treatment of the tumor.
Chapter • Gene Technology in Drug Research
Another technique analogous to proteomics is the
analysis of metabolites produced in an organism. The
term metabolome refers to all metabolites (e.g., metabolic
degradation products) present at agiven time. Metabolomics techniques attempt to quantify the metabolite
composition and use this information to infer the status
of acell. This is especially true when acell is exposed to
foreign substances. If the metabolite prole is studied at
aspecic time point, especially in pathophysiological or
genetically altered conditions, the term metabolomics is
used. The goal of this technique is to draw conclusions
about the molecular composition of cells from body uids such as urine, serum, or cerebral spinal uid. This
can lead to improved and more sophisticated diagnostic
procedures and, thus, to an easier early detection of diseases. It can also be used to characterize proteins for drug
therapy or to analyze the overall effect of adrug on cellular events. It is hoped that these techniques will lead to
abetter understanding of the overall effects of drug use
and ultimately to ahigher standard of safety in therapy.
12.10 Expression Patterns on aChip:
Microarray Technology
The analysis of the genome, transcriptome, proteome or
metabolome results in thousands of molecules that need to
be characterized. This ood of data requires an immense
measurement capacity. For this reason, the development of
microarray technology was initiated in the late 1980s. Thousands of molecules, which are to be analyzed in parallel in
an automated fashion, are attached to asupport made of
glass, silicon, gold, or nylon that is only afew centimeters
in size (. Fig.12.6). Very small quantities of biomolecules
are required. This technique is now sufciently mature to
be used in routine analytical procedures. In addition to
appropriate surface preparation, the art of reliable and
standardized immobilization of the molecules required for
precise analysis is critical for the success of the method. In
addition to proteins and protein domains, antibodies, antigens and especially DNA, oligonucleotides and RNA can
be immobilized. Proteins are often anchored by coexpress-
12
. Fig. 12.6
croarray technology. Individual gene segments are isolated from an organism and amplied by PCR (top left). They are then immobilized as
single-stranded oligonucleotides on amicrochip support (bottom left).
In addition to the isolated and amplied DNA, synthetically produced
DNA building blocks or cDNA molecules obtained by reverse transcription can also be immobilized on the support. One type of such
probe molecule is placed at each position on the support. RNA molecules are isolated from the cells of healthy (green) and diseased tissue
(red), transcribed into mRNA, and backtranscribed into cDNA (top
Producing and testing an expression pattern using mi-
right). The cDNA is labeled with auorescent dye. The test molecules
are then added to the microarray plate in asingle-stranded form, and
if they are complementary, hybridization will occur (bottom center).
Finally, the binding is analyzed under uorescent light (bottom right).
Yellow areas indicate that mRNA molecules from both healthy and diseased cells have bound. The mRNA that binds there is expressed in
both healthy and diseased states. Areas that remain dark indicate that
the mRNA is not upregulated in either the healthy or diseased state.
Areas that uoresce either only green or only red indicate adifference
in the expression pattern between cells from healthy and diseased tissue
. • SNPs and Polymorphism: What Makes Us Dierent
ing the protein of interest coupled to an anchoring protein
such as streptavidin as afusion protein. The streptavidin
anchor is attached to the surface via biotin. In addition,
thiol group chemistry is used. Disulde bridges are used
to couple the thiol groups to the surface, which has previously been provided with appropriate reactive groups.
Other strategies use amino groups, such as lysine, which
are then coupled to areactive aldehyde group on asolid
support. To test the composition of an analyte, asoluble
mixture is added to apremanufactured chip. If binding
partners are found in this transformation, the components
from the analyte solution will remain on the surface.
Such binding must be simple and detectable in aspatially resolved manner on the chip. Initially, staining and
uorescence were the methods of choice (. Fig.12.6).
Fluorescent dyes, such as green and red, are used because
they can be easily excited and detected in aspatially resolved way. If mixed signals occur due to simultaneous
red and green uorescence, ayellow signal is obtained.
In the meantime, surface plasmon resonance has become
more signicant (Sect.7.7). As an alternative, the latter
technique is used to detect binding. In addition, techniques similar to ELISA methods can be used (Sect.7.3).
Microarrays are often used to analyze the expression
patterns of biological systems. This is done by examining
the transcriptome of acell under different conditions, for
example, in adiseased and healthy state. The rst molecules
to be successfully anchored on chips were single-stranded
DNA oligonucleotides. To study the coding mRNA
of acell in aparticular state, these molecules are transformed into acomplementary DNA segment called cDNA
using areverse transcriptase (. Fig.12.6). These cDNA
molecules, or the fragmented sections of cDNA that are
obtained, are immobilized on achip and split into single
strands. The cell lysate containing the single-stranded
mRNA (transcriptome analysis) or the transcribed cDNA,
prepared from it, is added to such achip, and the complementary mRNA strand hybridizes with the oligonucleotide
fragments anchored there. It is important that the samples
to be analyzed are labeled with different uorescent dyes
depending on their origin. For example, the mRNA from
ahealthy cell is labeled green and that from adiseased cell
is labeled red. After hybridization on the chip, there are
areas that uoresce green, red, or yellow when excited, and
others that remain nonuorescent. Areas that uoresce
yellow indicate that mRNA molecules from both healthy
and diseased tissue are bound. Obviously, the mRNA that
binds there is equally available in either the diseased and
healthy states. Areas where no uorescence is seen indicate
that neither healthy nor diseased cells produced mRNA
that is bound there. Areas that uoresce either green or
red are of interest because they indicate differences in expression patterns between healthy and diseased cells. In
this way, gene products involved in adisease process can
be discovered. If there is dysregulation, drug therapy can
be used to try to correct the diseased state.
12.11 SNPs and Polymorphism:
What Makes Us Different
What makes an individual organism of a particular
species different and adds to the enriching diversity of
apopulation? We are talking about the human genome,
but there must be many interesting variations to make
us all look different and have different personalities.
Polymorphisms, or variations in the composition of the
genome, cause the observed diversity in or shape the different phenotypes of aspecies. The most obvious phenotypic difference is the division into male and female
individuals. Of course, this is not the only difference we
recognize in the human species. Many sequence variations occur within apopulation at the genome level. If
they occur in more than 1% of the population, they are
called different alleles, otherwise they are attributed to
mutations that have not yet become evolutionarily dominant. Genetic polymorphisms are observed, for example,
as insertions or deletions in which at least one nucleotide
is either partially or completely inserted or lost. The most
common sequence variation, however, is single nucleotide exchanges. The term SNPs (pronounced “snips”)
is used here, which is an abbreviation for single nucle-
otide polymorphism. Compared to the entire genome,
polymorphisms represent avery small fraction. They are
estimated to be 1% of the total genome, which is about
three million bases. Of these, SNPs are the overwhelming
portion, accounting for about 90%. Therefore, most of
our genome is identical across the human species, even
though there is enormous phenotypic diversity among us.
Within SNPs, coding and noncoding changes are distinguished according to whether the observed exchanges
are translated into proteins or not. In the coding regions
of the genome, asingle nucleotide exchange can lead to
an altered protein sequence. In Sect.32.7, the translation
process of abase triplet into aprotein is introduced. If
one base in acoding triplet is changed, the triplet can
either be translated into the same amino acid, or it can
lead to the incorporation of adifferent residue. This is
due to the fact that some triplets code for the same amino
acid. Incorporating adifferent amino acid into aprotein can change its properties. For example, the amino
acid composition of aglycosyltransferase determines the
blood group we have. An example of how achange in the
amino acid composition of aG protein-coupled receptor
can affect our sense of smell is given in Sect.29.7. Humans are divided into different alleles according to their
ability to sense different intensities and qualities of smell.
However, it is not only SNPs in coding regions that
lead to differences in our species. SNPs in noncoding
regions of the genome can lead to changes in gene regulation. In the context of drug discovery and therapy,
SNPs may be relevant even if they have no direct effect
on the phenotype. Some SNPs are thought to confer sus-
Chapter • Gene Technology in Drug Research
12
ceptibility to disease or inuence the cellular response
to adrug. It should be noted that SNPs may also occur
in the region of the binding site of adrug molecule,
which is not necessarily identical to that of the natural
substrate. SNPs then directly inuence the afnity and
binding prole of the drug. As aresult, adrug may exert
astronger or weaker inhibition of protein function in
patients with an observed SNP than in patients in whom
this SNP is not present.
12.12 The Personal Genome: Access to an
Individualized Therapy?
Genome sequencing and the analysis of SNPs and polymorphisms have impressively uncovered the source of
disease predisposition and why drugs have attenuated tol-
erability and different side effect proles. It has provided
an explanation for why undesirably large variations in
drug efcacy can occur between patients. All the more
reason to ask whether the sequencing of the individual
genome of each person would provide options for individual and personalized therapy. This is by no means
autopian idea, since it is now possible to sequence the
entire genome of an individual person in afew hours at
acost of less than US$ 1000.
In medicine, it is well known that the blood group of
the donor and the recipient must match for blood transfusions. Genome analysis would facilitate the search for
amatching donor organ for transplantation. Aparticularly high density of SNPs has been found in the genome,
especially in regions coding for proteins that present antigens to the immune system on their surface to stimulate
an immune response (Sects.31.7 and32.3). SNP analysis
of each individual could indicate the likelihood of developing aparticular disease. Early detection of this risk
and possible lifestyle changes could be better than any
therapy. Today, high-resolution DNA chips (Sect.12.10)
allow the simultaneous detection of more than 500,000
genetic SNP markers. Discovered SNPs can indicate
an increased predisposition to developing Alzheimer’s
disease in aging, for example. Asimple screening of an
individual’s DNA sequence could reveal apredisposition
to aparticular disease pattern.
Craig Venter, whose company sequenced the human
genome using the mRNA shotgun method, had analyzed
and published his own genome. The gene analyses of
these data revealed atendency towards obesity and cardiovascular disease. His own father had died of aheart
attack at the age of59. Based on this analysis, Venter
decided to take alipid-lowering drug from the statin
class as apreventive measure (Sect.27.3). Aphysician
could simply check the personal genome to see whether
the patient has aSNPs pattern that would indicate an
intolerance to aparticular drug therapy. In addition, the
physician could see what type of metabolizer category
(Sect.27.7) the patient belongs to. This could reduce intolerance to simultaneous treatment with several drugs
and allow safe adjustment of individual doses. It may
also help in choosing the right drug for atherapy, especially when several drugs with different modes of action
for one indication are available.
The dream of developing “personalized drug mole-
cules” for individual therapy will be difcult to realize
for cost reasons. Just adding one more methyl group to
adrug requires afull toxicological and pharmacological testing program to gain approval. It would consume
millions of dollars in development costs. As always, however, the determination of an individual’s genome and
the elucidation of all conceivable predispositions to possible diseases has its downside. In the hands of the treating
physician, this information is ablessing. But what would
afuture employer read into these data about the prospect
of hiring an employee? Insurance companies could accept only risk-free clients on the basis of their genomic
data—afrightening idea that an individual’s genomic
make-up would determine their insurance premium!
Regardless of our genetic differences and the possible consequences for drug therapy, we must not forget
that our gastrointestinal tract is home to millions of microorganisms. This ora has adecisive inuence on our
well-being, our health stability, our metabolism, and also
on our response to drug therapy. The individual gastro-
intestinal ora begins to build up at birth and is decisively inuenced by the mother. It varies considerably
with lifestyle, food culture, and exposure to the regional
microbial landscape. In India, China, or Europe you will
nd adifferent microbial culture than in America, for example. Interestingly, it changes when persons move their
homes between continents. Different microorganisms
cause adifferent conguration of secondary metabolites
and contribute to ashifted health balance. Presumably,
these differences between individuals are as important as
the genetic diversity that makes us different.
12.13 When Genetic Differences Turn into
Disease
Genetic diseases are molecular in origin. One gene (allele) is altered, sometimes the two genes from both parents. Each of us carries alarge number of such altered
genes, which are the result of random base exchanges:
the SNPs. The principle of evolution is based on these
random mutations. If amutation makes an individual
more adaptable to the environment, the chances of survival and reproduction will increase. These genes are then
reproduced with increased probability. In asexually reproducing species, horizontal gene transfer has an accelerated effect on evolution. Here, entire DNA fragments
are exchanged between individuals or even species. In this
sense, crossover plays an important role in sexual repro-
. • Epigenetics: Lifestyle and Environment Inuence Gene Activity Like aPen Leaves aM ark in the Book of Life
duction. In this case, adjacent gene sequences from both
parents randomly crossover and form new combinations.
Without mutation and crossover, all species would remain absolutely constant. In individual cases, many errors are produced as amechanism of evolution. Some of
these errors cause genetic diseases. In sickle cell anemia,
asingle amino acid in hemoglobin, which gives blood its
red color, is exchanged, and aglutamic acid at position5
of the beta chain of hemoglobinA (HbA) is replaced by
avaline. The modied hemoglobin aggregates: it “sticks”
together in the red blood cells. The cells collapse and take
on acharacteristic sickle shape. Homozygous carriers,
meaning individuals in whom the “sick” gene is inherited from both parents, cannot survive. Heterozygous
carriers, who carry one “sick” and one “healthy” gene,
produce both normal and altered hemoglobin. These
people have ashorter life expectancy, but usually reach
reproductive maturity. In areas where malaria is endemic,
there is aselection pressure for the genetic disease. Heterozygous carriers of sickle cell anemia are more resistant
to malaria than healthy individuals (Sect.3.2). Here we
are witnessing Nature’s great experiment. How will it
end? Even humans intervene. If malaria is successfully
treated, wild-type HbA carriers will no longer be at adisadvantage, and the evolutionary advantage of sickle cell
anemia and the resulting selection pressure towards this
disease will disappear. This genetic disease could become
“extinct” after afew generations. On the other hand, if
sickle cell anemia is treated either conventionally or by
gene therapy, then these people would have perfectly
normal “healthy” red blood cells. The malaria parasite
would be able to reproduce well in these cells again. The
protection against this disease would disappear, and the
susceptibility of these people to malaria would rise to
anormal risk level.
In addition to sickle cell disease, about four thousand
other diseases and their molecular causes are known.
Some, such as cystic brosis, phenylketonuria, and inherited coagulopathies, are relatively common. Many others
are rare, sometimes described only once. In recent years,
amultifactorial genetic cause has been identied for an
increasing number of diseases, including diabetes, rheumatoid arthritis, some cancers, asthma, and Alzheimer’s
disease. The occurrence of these diseases is caused by,
or at least facilitated by, the simultaneous occurrence of
multiple genetic changes.
The mechanisms of evolution are also responsible for
the development of resistance (Sect.3.2). In this case, the
selection pressure is exerted by adrug or an insecticide
(e.g., to eradicate malaria-carrying mosquitoes). Because
of their rapid reproduction, bacteria and viruses adapt
quickly to a“hostile” environment. The true masters are
retroviruses, which, due to their high mutation rate, can
develop resistance very quickly and, thus, wipe out the
success of adrug in one fell swoop (Sect.24.5).
12.14 Epigenetics: Lifestyle and
Environment Influence Gene
Activity Like aPen Leaves aMark in
the Book of Life
The development of an organism does not only depend on
the type of hereditary information stored in the DNA that
can be translated into gene products. It is just as important that certain genes are only transcribed in certain cells
at certain times. Social factors and the environment also
inuence genes and change their behavior. Scientists have
observed the following example in zebra nches. When
amale zebra nch hears the song of another male, the gene
EGR-1 is read more strongly. The unknown song of apotential rival leads to much stronger activity in EGR-1 than
background bird singing that the nch has already heard.
EGR-1 is itself akey gene in gene regulation, so achange
in the nch’s social environment leads to many shifts in the
bird’s protein expression pattern. This response helps the
male bird adapt to changing conditions in his environment,
as the intrusion of apotential competitor into his territory
can be of critical importance to him. The exciting question
is how these changes are expressed at the molecular level.
Pluripotent embryonic stem cells can differentiate into
many different cell types. For example, liver, brain and muscle cells have the same set of chromosomes, although they
are fundamentally different in function. Obviously, one
genotype gives rise to many different phenotypes. This is
true for the different cell types of an organism at the same
time, as well as for different developmental stages in an organism. Research on twins has yielded remarkable results
in this regard. Comparative studies of monozygotic twins,
who are genetically identical, show that with increasing
age, and especially with different lifestyles, there are progressively larger differences in phenotype. Therefore, there
must be mechanisms that lead to changes in the phenotype
that are passed on without changes in the genotype. They
regulate the transcription process and pass this feature on
to daughter cells. This process is called epigenetics. It leads
to the formation of an additional layer of information that
regulates the reading of genes from the DNA.
The environment affects genes through the epigenome.
Upbringing, childhood experiences, exposure to chemicals or intoxicants, and stress are all epigenetic regulatory
inuences that temporarily or even permanently alter gene
activity. As the following example of agouti mice shows,
such information can even be passed on to subsequent
generations. Normally, these rodents are small, brown,
slender, and very agile. Their genes contain the so-called
agouti gene, which, when activated, causes the animals to
become sick and their fur to turn yellow. Moreover, they
become greedy and fat. The offspring of these diseased
mice have exactly the same coloring and are just as fragile
as their parents. The American molecular biologist Randy
Jirtle at Duke University in Durham, North Carolina, fed
pregnant agouti females aspecial diet rich in supplements
Chapter • Gene Technology in Drug Research
12
. Fig. 12.7 The chromosomal DNA is shortened in its expansion by
afactor of 10,000 to 50,000 by coiling itself onto the basic histone
proteins, so that it ts into the cell nucleus as achromosome. The individual histones serve as akind of coil carrier (nucleosomes, diameter
about 110 Å). Approximately 150 base pairs t on such acoil. Many of
them line up like pearls on astring and fold in space to form chromosomes. Histone proteins are made up of helices. In the gure, positively
charged basic residues such as arginine and lysine are shown with blu-
ish surfaces, while negatively charged acidic residues such as aspartate
or glutamate are shown with reddish surfaces. The negative charges
of the DNA backbone, which contains many phosphate groups, are
such as vitamin B12, folic acid, choline, and betaine. As
aresult, the majority of these females’ offspring were
brown, slender, and in excellent health. The agouti gene
was switched off by the enriched diet without requiring
any changes to the rodents’ genome sequence.
At the molecular level, it is methylation and acetylation that carry the additional epigenetic information. In
contrast to genetic changes, which cause mutations in the
translated gene products, epigenetic changes have astrong
dynamic component and, above all, are reversible. In the
compensated by the many arginine and lysine residues. This creates
astrong electrostatic attraction. In order to read the DNA, this interaction must be loosened. For this purpose, individual Lys and Arg residues are acetylated, whereby the basic amino and guanidino groups
shed their positive charge. This causes adecrease in the compact binding of the DNA to the histone coils. (7 https://sn.pub/rpa861)
stretched state, there are more than two meters of DNA
in the cell, and this is wound in avery compact form on
small basic proteins: the histones. Lined up like pearls on
astring, they form the chromatin that makes up the chromosomes in their most densely packed form (. Fig.12.7).
Histones are the most highly conserved proteins in existence, for example, the 102-residue histone protein H4
from peas and cows differ in only two positions.
Epigenetic modications modify DNA by transfer-
ring methyl groups to cytosine by methyltransferases (see
. • The Scope and Limitations of Gene Therapy
Sect.26.10) to form 5-methylcytosine. Base pairing with
guanine in the DNA is not affected by this modication,
and the genetic code remains unchanged. When methylation occurs in apromoter region of DNA, it silences the
corresponding gene. The methylation makes the DNA
inaccessible to the reader, much like password-protected
computer data. If the promoters in these gene segments
are demethylated by methylases, translation into the cor
responding protein will be possible again. Asecond epigenetic modication is the modication of histone proteins. For example, histone acetyltransferases (HATs) can
enzymatically transfer acetyl groups to lysine residues of
these basic proteins. The added acetyl groups neutralize
the positive charge on the lysine residues (the so-called
“histone tails”). As aresult, they can no longer interact as efciently with the negatively charged phosphate
groups of DNA. Additional phosphate groups attached
to serine, threonine, and tyrosine residues by histone ki-
nases are even more repulsive. These changes result in less
densely packed chromatin, making it easier to read DNA
in certain regions. Transcription and gene expression are
regulated in this way. On the other hand, the cleavage
of acetyl groups by histone deacetylases (HDACs) or the
methylation of lysine and arginine residues of histones
by histone methyltransferases (HMTs) increases the pack-
ing density of chromatin and decreases the probability of
DNA transcription in affected regions.
The many proteins that create, recognize, or reverse
epigenetic changes are called writers (which make chemical modications to DNA and histones), readers (which
nd and interpret these modications), and erasers (which
remove the chemical marks as enzymes). Misregulation of
these enzymes is commonly associated with the development of various types of cancer. Since epigenetic processes are fundamentally reversible, there is apromising
opportunity to correct the dysregulated function of these
enzymes through drug therapy. In addition, attempts are
being made to manipulate the function of the protein
domains that read these modications. For this reason,
intensive research efforts are being devoted to nding ligands for the various methylases, methyltransferases, histone acetyltransferases, histone deacetylases, and reader
domains. The histone deacetylases that have been most
successfully studied to date are mechanistically closely
related to metalloproteases (Sect.25.10). It is hoped that
inhibitors of these enzymes, which induce or suppress disease-causing epigenetic changes, can be developed into
potent and safe drugs for human cancer therapy.
12.15 The Scope and Limitations of Gene
Therapy
In September 1990, 4-year-old Ashanti DeSilva became
the rst patient to receive gene therapy. Both of her parents had decient alleles for the enzyme adenosine deam-
inase. Because this enzyme is critical for the functioning
of the immune system, the little girl suffered from a severe immunodeciency that could not be treated with
conventional therapies. As a therapy, the patient’s white
blood cells were repeatedly infected with a virus that
carried the correct information for the missing enzyme.
The patient, who had previously been hospitalized and
-
was in constant danger of infection, became a person of
perfectly normal health.
Gene therapy refers to any technology that introduces
agene into apatient’s cell to replace adefective or miss-
ing gene. In principle, it is very simple. Viruses demonstrate it to us every day: they carry their own genetic
information into aforeign cell and use it to encode afew
key enzymes that are necessary for their own reproduction. For the rest, they use the biosynthetic machinery
of the infected cell. Retroviruses, whose genetic infor-
mation is encoded in RNA, translate this information
into DNA and integrate it into the host’s DNA. In gene
therapy, anucleic acid segment encoding the protein to
be replaced in the patient is inserted into the genome of
avirus. The construct, as these modied viral genes are
called, is surrounded by the viral capsid and introduced
into the patient’s cells. This can be done either outside
the body, such as in previously removed bone marrow or
white blood cells, or inside the body, such as by injection
into tumor tissue or aspecic organ.
Adenoviruses, herpesviruses, or retroviruses are all
well suited as gene carriers because these viruses incorporate their own genetic information into mammalian
DNA. Although retroviruses transfer their genes only
during cell division, adenoviruses can cause nondividing
cells to incorporate and use foreign genetic information.
Plasmids, DNA and liposomes, and pure DNA constructs are also being experimented with. The rates of
transfer of the new information into cellular DNA are
signicantly higher than is the case with viruses.
There are now more than 1000 gene therapy clinical
trials underway, most of them in the USA and mostly for
tumor therapy. Cancer is not an inherited disease, but the
genetic information that is passed from cell to cell creates
a“local genetic disease.” Oncogenes are alarge group
of proteins responsible for the development of cancer.
Tumor suppressor genes encode proteins that interfere
with the cell cycle and stop cells from dividing. The rapidly increasing knowledge of the molecular structure of
these proteins has opened up many approaches to gene
therapy of tumors.
Other diseases can also be targeted with gene therapy.
The standard therapy for cardiovascular diseases, which
are characterized by excessive growth of endothelial cells
and consequent narrowing of blood vessels, is to dilate
the vessels with aballoon catheter. This helps, but only
temporarily. After afew months, the cells start to proliferate again and the blood ow in the downstream areas
decreases threateningly. Gene therapy could be used here.
Chapter • Gene Technology in Drug Research
12
Adenoviruses can be released locally during the balloon
catheter treatment. These carry the genetic information
for aprotein that inhibits cell division, the so-called retinoblastoma protein. The cells are then unable to proliferate.
In 2014, the rst gene therapeutic drug, alipogentiparvovec, was approved for the treatment of hereditary
lipoprotein lipase deciency. This was followed in 2018
by voretigene neparvovec (Luxturna®), atreatment for
arare inherited retinal disease. Patients with hemophilia,
who require continuous replacement of missing blood
clotting factors, will soon benet from agene therapy
medicine. It will hopefully enable them to produce the
clotting factors in liver cells lifelong.
Will gene therapy replace traditional drug therapy?
The answer is denitely no. The technique is very complex and each patient needs an individually tailored therapy. Moreover, in many cases, results have been disappointing and sometimes devastating. Gene therapy will
take arm place in the treatment of certain diseases, as
it is acurative and not asymptomatic therapy. With increasing experience and better assessment of the possible
risks, interventions in the human genome will become
acceptable for some diseases, as they make it possible
to eliminate the genetic defect once and for all for the
individual and their offspring.
In recent years, genetic engineering, and with it the
possibility of gene therapy, has gained new momentum
from another direction. As early as in the 1980s, so-called
CRISPR sequences (clustered regularly interspaced
short palindromic repeats) were discovered in the ge-
nomes of various bacteria. These are repeated sequences
of 33–47 nucleotides that are interrupted by variable regions of other nucleotides. It is now known that these
sequences, in conjunction with the protein Cas, are used
to specically cut double-stranded DNA. This system,
known as CRISPR-Cas9, serves as an adaptive immune
defense for bacteria. For example, viral sequences introduced during aviral infection can be identied and
“registered”: in the event of anew infection with these
viruses, CRISPR-Cas9 can cut the associated DNA and
render it unusable.
Although this system originated from bacteria, it
functions as precise gene scissors in almost all organisms
and cells. The two scientists Emmanuelle Charpentier
and Jennifer Doudna recognized the potential of this
method for gene editing. It involves injecting acell with
the RNA that codes for the Cas9 protein and arecognition sequence. The cell uses this RNA to produce Cas9.
This protein then nds the enclosed recognition RNA
sequence. As aresult, Cas9 then cuts double-stranded
DNA according to the added RNA sequence fragment.
Thus, it is only the added RNA recognition sequence
that determines where Cas9 cuts the DNA. RNA fragments can be synthesized with any sequence, which theoretically means that the method can be used to cut at
any arbitrary but predetermined position within agiven
genome.
After the DNA is cut, there are separate pieces of
genetic material that are reassembled by cellular repair
mechanisms. This step is often imprecise and produces
short pieces of DNA (called “indels”) that can be inserted or deleted at the cutting site. This renders the
affected genes useless for further translation. Bacteria
use this very same mechanism in their adaptive immune
response to disable newly incorporated viral DNA.
However, gene editing uses a different step. If unbound DNA with loose ends is present in acell, it is
inserted without gaps into the cut DNA using the socalled HDR system (homology-directed repair). In this
way, the unbound DNA is introduced into the genome
as atargeted modication. As early as 2015, there were
reports about Chinese research groups using these genetic scissors to free human embryos from hereditary
diseases.
More recently, virus-based gene therapy and gene
scissors have been used in cancer therapy. In advanced
cancer, the body’s own gene-edited immune cells can
be used for therapy. The T-cells of the immune system
check whether acell has been transformed into acancer cell by degeneration (Sect.31.7). If such cells are
detected, they are normally eliminated by the immune
system. Unfortunately, degenerated cells often know
how to evade the immune response (Sect.31.7). To overcome this, white blood cells are taken from the cancer
patient and T-cells are isolated from them outside the
body. These T-cells are genetically engineered to carry
achimeric antigen receptor that specically recognizes
degenerate cancer cells. This can be achieved either by
introducing the modied DNA into the T-cells via viruses or by editing the genetic material of the T-cells
with genetic scissors. The result is genetically modied
CAR-T cells. They are then reinfused into the patient
(therapy with, for example, Kymriah® or Yescarta®),
where they recognize the cancer cells and initiate their
killing. The CAR-T cells multiply in the body, providing
long-term protection against the cancer.
The CRISPR-Cas9 system is currently the subject
of heated debate and is being studied for its applicability, but also for its risks and dangers for therapy. The
two scientists Emmanuelle Charpentier and Jennifer
Doudna were awarded the 2020 Nobel Prize in Chemistry for their achievement. Certainly, molecular biology
has never before had such precise and widely applicable
genetic scissors. But is its use fully understood, with
all the consequences for the modied organism? Will
there be long-term effects, and how will the newly introduced gene be transmitted to the next generation? One
can even ask whether an organism manipulated with
these genetic scissors has been genetically modied at
all. In fact, only anew variant of an already existing
genetic setup has been created. This happens over and