Chapter 2

Therapeutic Nucleic Acids 1

• This chapter describes in an analytical manner the
various types of molecules that are part of gene
therapy. These can be grouped into one of two classes:
• DNA sequences coding for proteins having various
cellular functions;
• nucleic acids (DNAs or RNAs) with regulatory function,
either synthesized as small synthetic molecules or, in the
case of RNAs, expressed inside the cells after transfer of
the corresponding genes.

2.1 Protein-Coding
• The concept of gene therapy was originally developed
with the idea of supplying a missing cellular function
by transferring a normal copy of the altered gene into
the relevant cells.

2.1 Protein-Coding
• In reality, in human cells the average size of proteincoding genes is 27 kb, by far longer than the maximum
length fitting the most common gene delivery systems.
• For this reason, gene therapy is most commonly based
on the transfer of cDNAs average length:
• 2.5 kb or of their protein-coding portion (average length:
1.5 kb, corresponding to about 500 codons.

2.1 Protein-Coding
• From the molecular point of view, the transfer of a
gene, or its cDNA or its cDNA coding region has
essentially different properties.
• Both cDNAs and their coding portions need to be
transcribed from promoters that are usually different
from the natural ones, which are commonly too large
to be used.

2.1 Protein-Coding
• In addition, the cDNA coding portions alone lack the
regulatory elements controlling gene expression at the
post-transcriptional level, which are usually contained in
the introns or in the untranslated regions (UTRs) at the 3'
and 5' ends of the cDNAs.
• These regions are commonly involved in the regulation of
• cDNA stability,
• transport,
• subcellular location and
• translation of the cellular mRNAs.

Protein-Coding Genes
• in several instances the levels at which proteins are produced
are not very important, and thus a tight translational or
posttranslational regulation of gene expression is not
• For example, this is the case of proteins replacing missing
cellular functions in the hereditary disorders of metabolism,
or antigens for anti-cancer vaccination, or secreted
• In these cases, transfer of the protein-coding region under the
control of a strong promoter, such as the promoter for the
cytomegalovirus (CMV) immediate-early (TE) genes, is

Protein-Coding Genes
• In several circumstances, inclusion of a small intron
upstream of the cDNA coding sequence, or of a 3' UTR
downstream of it, are known to facilitate expression of
the protein of interest.
• The proteins encoded by the therapeutic genes can
have very different functions, ranging from the
substitution of a missing cellular protein to the
modulation of the immune system.

2.1.1 Proteins Substituting Missing
or Mutated Cellular Proteins
• gene therapy was conceived with the purpose to express
proteins that are missing or defective, thus curing autosomal
recessive and X linked disorders.
• These replacement proteins can exert their functions
• inside the cells (for example, in the case of gene therapy for
muscular dystrophies) or
• on the cell membrane (for example, the CFTR gene in cystic
• or be secreted into the extracellular environment or the blood
stream (as is the case of coagulation factors in the hemophilias).

2.1.2 Proteins Modulating
Cellular Functions
• The objective of several gene therapy applications is
not to supply a missing cellular function, but to express
proteins able to modulate cell behavior.
• The approaches utilized are very variegate.

2.1.2 Proteins Modulating
Cellular Functions
• For example, in the cancer gene therapy field several
experimentations take advantage of the possibility to
induce arrest of cell proliferation using CDK inhibitors
such as p27 or p21, or checkpoint proteins such as p53.
• Again in the cancer gene therapy field, another
interesting class of proteins is those that increase the
therapeutic index of chemotherapy.

2.1.2 Proteins Modulating
Cellular Functions
• On several occasions, myeloid toxicity limits the dose
of antineoplastic drugs that can be administered to a
patient with a solid cancer.
• In these cases, it is possible to confer resistance to
CD34+ hematopoietic precursors by transferring into
these cells a gene coding for a membrane transporter,
such as the mdr gene, able to prevent the intracellular
accumulation of a large series of anticancer drugs.

2.1.2 Proteins Modulating
Cellular Functions
• Other examples of proteins modulating cellular
functions are those used for gene therapy of viral
infections, which block activity of some viral proteins
and thus impair viral infection.
• Among these proteins, RevM10 is a mutated form of
the HIV-l Rev protein, acting as a trans dominant
negative mutant:
• when this protein is present in CD4+ T lymphocytes, it
blocks wild-type Rev function and thus inhibits viral

• the immune response against cancer cells can
be increased by expressing, into these cells, the
genes coding for co-stimulatory proteins, such
as B7, ICAM-1, or LFA-3, which are commonly
downregulated in tumors as a mechanism of
immune escape and are however necessary for
proper antigenic presentation to cytotoxic T
lymphocytes (CTLs).

Cytotoxic T lymphocytes
• Lymphocytes that kill other ("target") cells.
• Targets may include:
• virus-infected cells
• cells infected with intracellular bacterial or protozoal
• allografts such as transplanted kidney, heart, lungs, etc
• cancer cells

2.1.3 Secreted Growth
Factors and Cytokines
• A number of gene therapy applications are based on
the delivery of genes coding for secreted factors and
cytokines, having various activities.
• For example, in cancer gene therapy, several clinical
approaches take advantage of the genes coding for
interleukin-2 (IL-2), lL- 12, IL-7, IL-4, GM-CSF, and
other cytokines to increase antigen stimulation or
modulate the immune response against the
transformed cells.

2.1.3 Secreted Growth
Factors and Cytokines
• In the field of cardiovascular disorders, vascular
endothelial growth factor VGF gene transfer is used by
a number of applications aimed at the induction of
therapeutic anginogenesis in patient with cardiac or
peripheral iskemia, due to the powerful activity of this
factor in inducing new blood vessel formation.

2.1.3 Secreted Growth
Factors and Cytokines
• Finally, gene therapy for a number of
neurodegenerative disorders is based on the delivery of
genes coding for neurotrophic factors, including
• nerve growth factor (NGF) in Alzheimer's disease,
• neurturin (NTN) in Parkinson's disease and
• ciliary neurotrophic factor (CNTF) in Huntington's



2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• Several pathologic conditions are caused by the death
of some cell types or, on the contrary, by inappropriate
survival of others.

2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• Examples of the former condition are
neurodegenerative disorders, which are caused by
accelerated apoptotic death of some neuronal
• in contrast, tumors represent a paradigmatic example
of a condition in which lack of cell apoptosis essentially
contributes to disease development.

2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• Since apoptosis ensues as the net result of the function
of various proteins favoring or contrasting this process
• transfer of the genes coding for these proteins might
have a therapeutic role.

2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• For example, one of the gene therapy approaches for
amyotrophic lateral sclerosis (ALS), a motoneuron
disorder, is based on the transfer of the hc/-1 gene,
coding for an anti-apoptotic protein.

2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• In contrast, apoptosis in tumors can be induced by
oligonucleotides targeting a few antiapoptotic proteins,
• Bcl-1 itself,
• Survivin, or
• the X-linked inhibitor of apoptosis (XIAP).

2.1.4 Proteins Regulating
Cell Survival and Apoptosis
• Another way to interfere with cell survival is the socalled "suicide gene" approach.
• Cells are transduced with a vector expressing the
thymidine kinase gene of the herpes simplex virus type
1 (HSV-TK), an enzyme that is innocuous per se but
becomes toxic in the presence of the drug gancyclovir.
• The result of this activation is a block in DNA synthesis
and consequent cell death by apoptosis.

2.1.5 Antigens for

2.1.5 Antigens for
• A vast range of gene therapy applications have the
immune system as their target.
• These consist in the utilization of gene transfere to
activate the immune system, by either the delivery of
• genes coding for immunomodulatory cytokines or
• transferring, into cancer cells, genes coding for the coimmunostimulatory proteins necessary for antigenic

2.1.5 Antigens for
• A growing field of gene therapy applications in this are
is that of DNA-based vaccination (genetic vaccination).
• This is based on the in vivo delivery of genes usually
coding for viral or tumor cell antigens, to seek
activation of the immune system against the encoded

2.1.5 Antigens for
• The antigen gene can be transferred using naked
plasmid DNA "DNA vaccination" sometimes using
physical methods such as
• gene transfer facilitators (for example, the "gene gun"
approach), or
• viral (adenovirus, vacciniavirus)

2.1.5 Antigens for
• Genetic vaccination offers several advantages over the
common vaccination strategies based on the
administration of either attenuated or inactivated
microorganisms, or on protein antigens.

2.1.5 Antigens for
• These include the possibility to evoke a cytotoxic
immune response, since the processed exogenous
proteins are directly expressed in the context of MHC
Class I, together with the
• relative safely,
• reduced cost, and
• prolonged storage.

• Cancer and HIV-1 infection are among the disorders in
which genetic vaccination is most commonly considered.

2.1.6 Antibodies and
Intracellular Antibodies
• A peculiar class of therapeutic genes are those coding
for antibodies.
• Natural antibodies have a typical Y-shaped structure,
composed of 4 polypeptide chains: two identical heavy
(H) chains ( -440 amino acids each) and two identical
light (L) chains (- 220 amino acids).
• Both the H and L chains contain a variable region V (V
H and V L), which together recognize the antigen.


2.1.6 Antibodies and
Intracellular Antibodies
• The H chains then contain three constant (C) regions
(CHI, CH2, and CH3) and a flexible “hinge” (h) region
between CH 1 and CH2, while the L chains only have
one constant region (CL)
• The H and L chains are synthesized separately in the
B-cell endoplasmic reticulum and assemble thanks to
the formatton of both inter- and intra-chain disulfide

2.1.6 Antibodies and
Intracellular Antibodies
• Digestion of an antibody molecule with the proteolytic
enzyme papain generates three fragments, which can
be separated by chromatography.

2.1.6 Antibodies and
Intracellular Antibodies
• Two of these are identical and
correspond to the antigenbinding portion of the antibody
(antigen-binding fragment,
Fab), composed of the whole L
chain (VL +CL) and the Nterminal portion of the H chain,
including the variable region
(VH) and the CH1 constant

2.1.6 Antibodies and
Intracellular Antibodies
• The third fragment (crystallizable
fragment, Fc) is instead only composed
of the C-terminal region of the H chain,
which is able to bind complement and
different receptors,
• An Fc receptor is a protein found on
the surface of certain cells - including

B lymphocytes,

natural killer cells,



mast cells - that contribute to the protective
functions of the immune system.

2.1.6 Antibodies and
Intracellular Antibodies
• The fact that an antibody consists of four polypeptide
chains poses obvious problems for its expression by
gene transfer.
• However, it is possible to obtain synthetic antibodies
consisting of a single polypeptide chain formed by the
V portions of the H and L chains (Vh and VL) of the
natural antibodies, separated by the flexible amino
acid linker

2.1.6 Antibodies and
Intracellular Antibodies
• This construct, named single-chain Fv (scFv), contains
all the structural determinants allowing specific
binding of the molecule to its target antigen.
• While the mass of a natural IgG antibodys about 150
kDa, that of a scFv is 29 kD, corresponding to about
250 amino acids.

2.1.6 Antibodies and
Intracellular Antibodies
• The Vh and VL regions contain three hypervariable
regions that essentially participate in antigen
recognition (CDR).
• An even simpler form of antibody only contains the
CDR regions of one chain.

2.1.6 Antibodies and
Intracellular Antibodies
• These are named single domain antibodies, with
analogy to the antibodies produced by some camelids,
in which the antigen-binding site is composed of a
single chain, analogous to the mammalian H chain.

2.1.6 Antibodies and
Intracellular Antibodies
• Neither scFv nor single-domain antibodies
exert any effector activity (compment fixation, binding
to cellular receptors), since these are commonly
associated with the Fc portion of natural
• The simplest manner to obtain an scFv
antibody is to clone the Vh and VL region of a
monoclonal antibody (mAb) having high
affinity for the antigen of interest.

2.1.6 Antibodies and
Intracellular Antibodies
• Alternatively, the scFv can be obtained by the direct
screening of an scFv library, for example a phage
display library, in which the antibody is displayed on
the phage surface.
• The scFv-coding gene can thus be cloned into a
plasmid or a viral vector and transferred into the cell.

2.1.6 Antibodies and
Intracellular Antibodies
• When the scFv gene coding sequence is preceded by a
secretion signal, the antibody is secreted outside the
• In order to stabilize an scFv antibody and increase its
avidity the VL-VH region can be followed by a portion
of the antibody Fc fragment.
• This can induce dimerization of two scFvs and,
formation of disulfide bonds.
• These types of engineered antibodies are known as

2.1.6 Antibodies and
Intracellular Antibodies
• Natural antibodies are secreted in serum or expressed
on the surface of B lymphocytes and recognize their
target antigens only when these are present in the
extracellular environment or expressed on the cell
• In the absence of a secretion signal, the scFv antibodies
can instead be translated in the cytosol.

2.1.6 Antibodies and
Intracellular Antibodies
• In this reducing compartment, disulfide bonds cannot
be formed, however some scFvs can still fold properly
in the absence of these bonds and retain their binding
• These intracellular antibodies, also named intrabodies,
thus have the important potential to target
intracellular antigens.

2.1.6 Antibodies and
Intracellular Antibodies
• Both scFv and single-domain intracellular antibodies can be
used for a vast series of therapeutic applications.
• They can block interaction between two intracellular
proteins, or protein binding to DNA, or inhibit function of
an enzyme.
• Alternatively, they can re-localize a protein to a cellular
compartment different from its normal, thus blocking its
• For example, intracellular antibodies having a nuclear
localization signal can bind a cytosolic antigen and relocalize
it into the nucleus.

2.1.6 Antibodies and
Intracellular Antibodies
• A vast series of pre-clinical studies have shown the
potential efficacy of intracellular antibodies for gene
therapy of various disorders.
• In the field of gene therapy for viral infections, the cell
can be rendered resistant to infection by intracellular
antibodies blocking function of the proteins, of either
viral or cellular origin, that are essential for viral

2.1.6 Antibodies and
Intracellular Antibodies
• In gene therapy of cancer, intracellular antibodies are
used to modify the intracellular localization, and thus
inhibit the function, of oncogenes such as ErbB-2 in
breast and ovary carcinoma.

2.1.6 Antibodies and
Intracellular Antibodies
• In the field of gene therapy for neurodegenerative
disorders, the use or intracellular antibodies can block
the accumulation of pathological proteins, such as
• Tau in Alzheimer’s disease or
• the PrPsc protein in prion disease.

2.1.7 T-Cell Receptor
(TCR) Subunits
• An interesting class of protein-coding genes is that
including variants of the T-cell receptor (TCR), used to
modify the target specificity of T-lymphocytes.
• In both viral infection and cancer a potentially
powerful therapeutic approach would consist in the ex
vivo activation of the patients CD8+ CTLs reacting
against specific viral or tumor antigens, followed by
their reinfusion in vivo, a procedure known as adoptive

2.1.7 T-Cell Receptor
(TCR) Subunits
• More efficacious than recovering the endogenous
antigen-specific CTLs, which are usually limited in
number, this can be achieved by modifying the
specificity of any CTL by transducing these cells with
the genes coding for a TCR of choice.
• The TCR consists of a complex of at least 6 different
• Antigen recognition specificity is conferred by a
heterodimer of a/b (in most cases) or g/d chains.

T-Cell Receptor (TCR) Subunits

2.1.7 T-Cell Receptor
(TCR) Subunits
• The simplest manner to confer TCR specificity to T lymphocytes
is to transfer, into these cells, the genes coding for the a and b
chains specific for an antigen of interest obtained from a
previously selected natural cell clone.
• Gene transfer is commonly achieved using a retroviral vector in
which the genes coding for the two chains are separated by an
• A potential limitation of this approach, however, is that the
exogenously introduced TCR molecules might complex with
those endogenously expressed by the T cells, thus generating
TCRs with different, and potentially undesired, target specificity.

2.1.7 T-Cell Receptor
(TCR) Subunits
• An effective manner to overcome these problems is to
exploit alternative ways to modify TCR specificity.
• One of these consists in the use of single-chain TCRs,
in which mispairing between a and b chains is
prevented. Another possibility is offered by the so
called T-bodies.

2.1.7 T-Cell Receptor
(TCR) Subunits
• These consist of a genetic fusion
between a single-chain antibody
recognizing the antigen of interest
and the TCR CD3 zeta chain, able
to activate the signal transduction
cascade leading to cell activation.
• This approach both renders the
engineered CTL independent from
MHC restriction and prevents
mispairing between the exogenous
and endogenous TCR a and b