Chapter 2

Therapeutic Nucleic
Acids
II

2.1.8 Control of Therapeutic Gene

Expression
As already mentioned above, several of the
current gene therapy applications based on the
intracellular expression of protein-coding genes
take advantage of strong constitutive
promoters, such as the CMV IE gene promoter.

2.1.8 Control of Therapeutic Gene

Expression
In several situations, however, expression of
the therapeutic protein (or regulatory RNA)
must be
limited to one specific cell type,
or at least controlled in terms of levels,
or restricted to a defined temporal period.
For example, the genes coding for the different
hemoglobin chains (alpha-and beta-globin in
the adult) must be transcribed in a balanced
manner

2.1.8 Control of Therapeutic Gene

Expression
To avoid monomer aggregation and
precipitation in the erythroblasts, causing
apoptosis of these cells
in gene therapy of diabetes, insulin must be
precisely produced in response to blood
glucose concentration.
Expression of some pro-apoptotic genes can be
beneficial only if limited to cancer cells.

2.1.8 Control of Therapeutic Gene

Expression
over-expression of some growth factors,
including those inducing new blood vessel
blood formation, might be detrimental
expressed for excessively long periods.

2.1.8 Control of Therapeutic Gene

Expression
In several of these conditions, an optimal
solution would be to use the natural promoter
of the therapeutic gene to direct its expression
in vivo.
In most cases, however, this is not possible,
since natural promoters are usually very large
and thus cannot be accommodated in most
viral vectors.

2.1.8.1 Tissue-Specific Promoters

The first strategy to tackle the issue of
regulated trans gene expression is to limit its
transcription to the tissue of interest, using a
promoter specific for this tissue and sufficiently
short to be cloned in a gene transfer vector.

2.1.8.1 Tissue-Specific Promoters

Examples of such transcriptional elements are
the musde creatine-kinase (MCK) enhancer
the beta-actin gene promoter for the skeletal
muscle,
the a-myosin heavy chain gene promoter
(alpha MHC) for the heart,
the insulin gene promoter for the pancreas,
the albumin gene promoter for the liver,
the transthyretin gene promoter for the
retina, and so on.

2.1.8.1 Tissue-Specific Promoters

An elegant manner to obtain the opposite
effect, namely block transgene expression in a
given tissue, is to act post-transcriptionally and
exploit the presence, in several cell types, of
natural microRNAs targeting various cellular
genes .

2.1.8.1 Tissue-Specific Promoters

If the trans gene mRNA sequence is the target
of a miRNA which can be obtained by cloning
the miRNA recognition sequence downstream
of the transgene coding region and before the
polyadenylation site- protein expression will be
selectively inhibited in the cell types
expressing this miRNA while remaining
unaffected in other cells.

2.1.8.1 Tissue-Specific Promoters

This property can be used, for example, to
block expression of a protein of interest in
antigen-presenting cells (APCs), thus blocking
its presentation to the cells of the immune
system.

2.1.8.2 Inducible Promoters

Besides the use of tissue-specific promoters, an
alternative manner to regulate therapeutic
gene expression is by using inducible
promoters, in which transcription can be
selectively activated.
Some of these promoters are naturally present
in the genome and direct gene expression
under specific physiologic conditions.

2.1.8.2 Inducible Promoters

Among these promoters are those of genes
expressed
in response to heavy metals- for example the
methallothioein gene,
heat shock proteins 70 and 90,
hormones
MMT Virus long terminal repeat (LTR) promoter,
sensitive to dexamethazone induction,
and hypoxia -for example the VEGF angiogenic
protein.

2.1.8.2 Inducible Promoters

Most of these are natural promoters, however,
cannot be easily used since transcriptional
control is not very stringent,
the level of induced expression are too low,
they are activated in conditions that cannot
be easily reproduced in a therapeutic setting,
the pleiotropic undesired effects elicited by
the stimulating agent itself (for example, high
temperature or hormone administration).

2.1.8.2 Inducible Promoters

A very interesting category of promoters, in
contrast, consists of a few synthetic promoters,
the activity or which can be controlled
phamacologically, by the administration of
simple drugs.

2.1.8.2 Inducible Promoters

These promoters respond to specific
transcriptional activators that can be assigned
to one of at least three classes:
1. transcriptional activators regulated by small
molecules;
2. intracellular steroid hormone receptors; or
3. synthetic transcription factors in which
dimerization is controlled by antibiotic
rapamycin.

Inducible Promoters

transcriptional activators controlled by small

molecules
This class of inducible regulators is based on the
use of transcription factors that change their
conformation (and are thus either activated or
deactivated) upon binding one small chemical
molecule.
The prototype of this class of regulators is the Tet
repressor (TetR), which, in E.coli regulates
expression of the Tn10 operon genes.
This operon encodes a system of transporters that
determine exit of tetracycline from the bacterial
cell, thus conferring resistance to this antibiotic.

2.1.8.2 Inducible Promoters

transcriptional activators controlled by small

molecules
ln the absence of tetracycline, TetR binds a
DNA sequence (the Tet operator, tetO)
positioned upstream of the Tet operon and
suppresses transcription.
When tetracycline is instead present, this binds
TetR and causes a confonnational change that
blocks its interaction with tetO: in this manner,
transcription of the Tn10 operon is derepressed and the bacterium becomes resistant
to the antibiotic.

Inducible Promoters

transcriptional activators controlled by small

molecules
This transcriptional control strategy was adapted to
mammalian cells by generating a two-component system:
on one hand, the gene of interest is placed under the
transcriptional control of a minimal promoter - which
dictates the localization of the transcription start site- with a
series of TetO repetitions positioned upstream;
on the other hand, another construct codes for a fusion protein
between TetR and an RNA polymerase II transcriptional
activator, such as the carboxyterminal domain of the herpes
simplex virus type I (HSV-1) protein VPJ6 (the fusion protein
between TetR and VPI6 is known as tTA).
In the absence of tetracycline, tTA binds in multiple copies to
the promoter and activates transcription (Fig. 2.3A).

Inducible Promoters

transcriptional activators controlled by small

molecules
When tetracycline or, better, its derivative
doxycycline is present, transcription is instead
switched off since the antibiotic binds the TetR
moiety of tTA and the allosteric change
determined by this interaction causes
detachment of the hybrid transcription factor
from the promoter.
This system of transcriptional regulation is
known as Tet-off; since the antibiotic blocks
transcription.

Inducible Promoters

transcriptional activators controlled by small

molecules
A variant of this system was subsequently
developed, based on the use of a four-aminoacid mutant of the TetR protein, known as
reverse TetR (rTetR), which is unable to bind
the promoter unless the antibiotic is present.
When this protein is fused to VP16 to generate
the reverse tTA (rtTA) regulator, transcription is
switched on by the addition of the antibiotic
(Tet-On system; Fig. 2.3B).

Inducible Promoters
transcriptional activators controlled by small
molecules
A couple of these are derived from E. coli and exploit
the antibiotic- dependent binding of other
activators/repressors to their cognate DNA sites; these
include
the PIP transactivator binding the PIR binding site,
which is regulated by pristinamycin, and
the E transactivator binding the ETR binding site,
regulated by erythromycin.
Alternatively, a system regulated by gaseous
acetaldehyde was also developed, based on the AlcR
transactivator derived from the fungus Aspergillus
nidulans.

Intracellular receptors for steroid

hormones
Intracellular receptors are proteins that, once
complexed with their ligands, directly activate target
gene expression in the nucleus.
The prototypes of this class of molecules are the
steroid hormone receptors.
In particular, the estrogen and progesterone receptors
are sequestered in the ceII cytosol by the Hsp90
protein, which masks their nuclear localization signal;
binding to the respective hormones induces a
conformational change that releases the receptors,
allows their nuclear import, and thus activates target
gene transcription.

Intracellular receptors for steroid

hormones
To exploit this system for the inducible expression
of a desired gene, the natural receptor protein was
engineered by fusing it with the DNA binding
domain of the yeast transcription factor Gal4,
in order to target this fusion protein towards
synthetic promoters containing Gal4 binding sites,
and by mutating its amino acid sequence to lower
its affinity for the natural hormones and increase
that for synthetic analogues, including estrogen
and progesterone receptor.

Intracellular receptors for steroid

hormones
When cells are transfected with a constract
containing the gene of interest under the
control of the promoter containing the Gal4
binding elements and another construct
expressing the engineered receptor,
transcription of the gene of interest can be
selectively controlled by administering the
synthetic hormone

Intracellular receptors for steroid

hormones
A peculiar type of steroid hormone receptor is
the D. melanogaster ecdysone receptor (EcR).
In insects, the steroid hormone ecdysone has
an essential role in stimulating
metamorphosis.
The hormone acts by entering in the cells and
binding a receptor composed of hetrodimer of
two proteins, the EcR itself and the
ultraspiracle (USP) gene product

Intracellular receptors for steroid

hormones
In this manner, expression of the gene of interest only
occurs after administration of ecdysone or its synthetic
analog muristeron A or ponasteron A.
In mammals, the pharmocokinetics of these hormones
is very favorable, since
they neither accumulate
nor are eliminated too rapidly,
are not toxic and
are inactive in the absence of their cognate receptors.
Furthermore, the lipophilic nature of these compounds
allows them to freely cross the plasma membrane and the
blood-brain barrier.

Transcriptional control by ligand-induced

transcription factor dimerization
A third system to achieve
pharmacologically induced transcription
is based on the property of the drug
sirolimus (rapamycin, a macrolide antibiotic
originally extracted from a Streptomyces)

Transcriptional control by ligand-induced

transcription factor dimerization
This drug has been used for over a decade as an
immunosuppressant in transplanted patients and
is now also being tested for cancer therapy.
Inside the cells, rapamycin interacts with a
member of the FKBP (FK506-binding protein)
family, the immunophilin FKBP12.
The rapamycin/FKBP 12 complex then binds a
specific domain (the FRB domain) of mTOR
(mammalian target of rapamycin, a protein of the
PBK family) protein kinase and blocks its action.

Transcriptional control by ligand-induced

transcription factor dimerization
Since this kinase is essential for signal
transduction in T cells, rapamycin causes cell
cycle arrest in the Gl phase of the cell cycle;
lack of T-cell proliferation explains the
immunosuppressive effect of the drug.
Rapamycin is thus the prototype of a drug
selectively inducing dimerization of two
proteins, FKBP12 and mTOR.

Transcriptional control by ligand-induced

transcription factor dimerization
When FKBP is fused to the zinc linger (ZF)
domain of a transcription factor and its FRB
partner to a transcriptional activation domain,
the presence of rapamycin induces the
formation of a heterodimeric transcription
factor;
this synthetic factor binds a specitic target
sequence thanks to the ZF domain and
activates transcription through its activation

 transcription can be activated over 10,000-fold

in the induced condition compared to the basal
level.
However, they require that, together with the
therapeutic gene, the gene coding for the
inducible regulator is also transferred into the
same cell.

 In addition, the regulator often consists of two

different proteins, as in the case of the
ecdysone- or rapamycin-regulated systems.
This poses important limitations to the clinical
experimentation, since it obliges the
experimenter to accommodate several
transcriptional cassettes within the same
vector, and to use vectors allowing cloning of
large DNA inserts.

 ln addition, the regulatory proteins must be

expressed in a constitutive manner; however,
these proteins derive from different organisms
and are thus potentially immunogenic.
Finally, the above-described inducible systems
can efficiently regulate RNA polymerase II
transcription, while they are difficult to adapt to
RNA polymerase lll regulation, which, by its
own nature, tends to be constitutively active.

 Thus, the inducible systems are effective in

regulating protein- and microRNA-coding
genes, but much less the genes coding for
shRNAs or other small regulatory RNAs

2.2 Non-Coding Nucleic Acids

Besides protein-coding nucleic acids, the spectrum or gene
therapy applications is enormously increased by the
possibility or using short nucleic acids (DNAs or RNAs) with
regulatory function. These molecules belong to one of at
least six possible classes:
oligonucleotides and modified oligonucleotides;
small catalytic RNAs and DNAs (ribozymes and DNAzymes)
small regulatory RNAs (siRNAs and microRNAs);
long antisense RNAs;
decoy RNAs and DNAs;
RNAs binding to other molecules thanks to their
tridimensional structure (aptamers).

 DNA oligonucleotides and modified oligonucleotides

must be administered to the cells from the outside.
In contrast, the RNA molecules can also be synthesized
inside the cells by transferring their coding DNA
sequences.
all steps regulating gene expression can be controlled by
small regulatory molecules, including
transcription and splicing (oligonucleotides),
mRNA decay (oligonucleotides, ribozymes, DNAzymcs,
siRNAs and long antisense RNAs),
protein synthesis (siRNAs), and
protein function (aptamers and decoys).

2.2.1 Oligonucleotides
The simplest form of nucleic acids with
potential therapeutic function consist in short,
chemically synthesized, single-stranded DNA
molecules, usually 15-100 nt long.
Use of these oligodeoxynucleotides is based on
the intrinsic property of a DNA strand to pair
with its complementary sequence thanks to the
formation of hydrogen bonds between the
nucleotide bases.

2.2.1 Oligonucleotides
Although each of these bonds is non-covalent
and thus weak, their overall number renders
the overall affinity between two
complementary DNA
Furthermore, base pairing is extremely specific
in terms of target recognition, since a 17-nt
DNA stretch is statistically present only once in
the entire human genome.

2.2.1 Oligonucleotides
synthetic oligodeoxynucleotides can be used in at least four
different types of applicalions: the first two targeting cellular
mRNA and the second two genomic DNA;
1. to block gene expression, exploiting base paring between
the oligodeoxynucleotides and a target RNA sequence;
2. to modulate pre-mRNA splicing, thus favoring or impeding
inclusion of one exon in the mature mRNA;
3. to block transcription, by promoting formation of triple
helix DNA structures, usually in the promoter region of a
target gene;
4. to promote gene correction, exploiting
oligodeoxynucleotide pairing with a homologous genomic
DNA segment.

Antisense oligodeoxynucleotides
(ASOs) blocking gene expression
To selectively inhibit expression of a cellular or
viral gene, 17-22-nt oligodeoxynucleotides can
be used, having a sequence complementary to
that of the target mRNA.
Once introduced into the cell, pairing of the
ASO with its target blocks ribosomal translation
and stimulates degradation of the RNA:DNA
hybrid by cellular RNase H enzymes.

Antisense oligonucleotides (ASOs) that

modulate splicing
Thanks to its complementarity to target mRNA,
an ASO can also be used to induce exclusion of
an exon during premRNA maturation.
ln some diseases, presence of a point mutation
in an exon can generate a stop codon, leading
to the synthesis of a prematurely truncated
protein, or shifting the open rending frame
downstream of the mutation.

Antisense oligonucleotides (ASOs) that

modulate splicing
In some situations, such as Duchenne muscular
dystrophy (DMD), the disease caused by such
mutations is much worse than that eventually induced
by the absence or the entire exon containing the
mutation.
One strategy to induce exon skipping from an mRNA
is to treat cells with an ASO complementary to the
signals that regulate pre-mRNA splicing.
These ASO typically target the 5' and 3' splice sites or
the sequences internal to the exon that determine its
retention in the mature mRNA (exon sequence
enhancers, ESEs).

Triple helix forming

oligodeoxynucleotides (TFOs)
TFOs are single-stranded oligodeoxynucleotides
binding the DNA major groove in a sequencespecific manner.
In particular, they form very stable bonds with
a DNA duplex of 10-30 bp, containing a stretch
of purines and a complementary stretch of
pyrimidines.
The third helix is formed by the TFO, which,
according to the target sequence, can be made
of either purines or pyrimidines.

Triple helix forming

oligodeoxynucleotides (TFOs)
The TFO binds the target duplex through the
formation of two hydrogen bonds between
each of its bases and the purine-rich strand of
the target DNA.
Formation of a triple helix is thus an intrinsic
property of DNA structure and does not require
any cellular protein.

Triple helix forming

oligodeoxynucleotides (TFOs)
When the TFO target is in the promoter of a
gene, or immediately downstream of the
transcription start site, the triple helix structure
impair transcription factor binding or duplex
DNA unwinding, thus blocking gene expression.

Triple helix forming

oligodeoxynucleotides (TFOs)
In some instances, formation of a triple helix
was also shown, although at low efficiency, to
promote DNA repair, thus inducing the
correction of mutations, by recruiting the
cellular machineries responsible for excision
repair or homologous recombination.

Oligonucleotides inducing correction of point

mutations
One of the most ambitious goals of gene
therapy is to directly modify the genetic
information to obtain the correction of a
pathological mutation.

Oligonucleotides inducing correction of

point mutations
This objective can be met by introducing, into
the cells a DNA stretch having a sequence
identical to the region to be corrected, however
without the mutation, and then exploiting the
cellular machinery involved in DNA repair for
the substitution of the genomic DNA sequence
with that administered exogenously.

Oligonucleotides inducing correction of point

mutations
gene conversion process is usually based on
the cellular machinery responsible for
homologous recombination, and uses, as
substrates, long, single-stranded DNA
stretches, composed of several thousand
nucleotides.

Oligonucleotides inducing correction of

point mutations
In some cases, however, short, single-stranded
oligonucleotides, composed of 20-110 nt and
having mismatched nucleotides in their central
position, can stimulate gene correction by
favoring the recruitment of cellular DNA
mismatch repair protein.
Although this approach is experimentally
interesting, the fequency at which gene
correction can be obtained is usually very low
(usually less than O.l% of the treated cells) and
still restricted to cultured cells.

2.2.2 Modified Oligonucleotides

Among the essential problems limiting
application of oligodeoxynuclcotides in vivo is
their
limited tissue distribution,
cytotoxicity, and
low stability.

2.2.2 Modified Oligonucleotides

Both in the extracellular environment and
inside the cell, short natural DNA molecules, in
which nucleotides are connected by
phosphodiester bonds, are rapidly degraded by
endo- and exonucleases (DNases).
To overcome this problem and thus increase
bioavailability of these molecules in vivo, the
oligodeoxynucleotides structure can be altered
by introducing various chemical modifications

Modified Oligonucleotides
first-generation
In first-generation modified oligonucleotides,
one non-bonding oxygen atom in the
phosphate group is substituted by a sulfur
atom (phosphorothioate
oligodeoxynucleotides).
This modification confers higher stability to the
molecules and thus increases their half-life.

Modified Oligonucleotides
first-generation
In vitro, phosphorothioate oligodeoxynucleotides can
be efficiently delivered to cells using lipofection.
In vivo, they can be used in the form of naked
molecules, however their half-life is very short (less
than two hours in serum and four hours in tissues)
and therefore they need to be administered by
continuous intravenous infusion.
Phosphorothioate oligodeoxynucleotides are
relatively well tolerated, with minimal side effects
probably due to the interaction of the polyanion
backbone with some serum proteins.

Modified Oligonucleotides
second generation
A second generation of modified
oligonucleotides was obtained by the
introduction of an alkyl group in the 2' position
or the ribose molecule of nucleotides.
The generated molecules include '2'-0-methyand '2'-0-methoxyethyl-RNA.
These molecular species are less toxic than
phosphorothioate oligodeoxynucleotides.

Modified Oligonucleotides
second generation
The conformation changes induced by these
modifications and, in particular, the presence
of ribose, improve target binding, however they
also abrogate the oligonucleotide:target mRNA
duplex ability to activate RNase H, a crucial
aspect of ASO activity.
Thus, the inhibitory effect of these molecules is
only exerted through the inhibition of mRNA
translation and is thus lower than that of
phosphorothioates.

Modified Oligonucleotides
second generation
This drawback can be avoided by generating
chimeric oligonucleotides with 2'-modified
nucleotides placed only at the ends of the
oligonucleotide, thereby leaving a central
RNase-compatible DNA gap.
These hybrid oligonucleotides are known as
gapmers.