Delivering Small interfering RNA or si-RNA molecules in-vivo to treat diseases. Definitions, Approaches, Barriers, Solutions, Delivery systems, and more. With an overview on gene delivery systems and philosophy.

1. Gene Delivery Slide 1 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
siRNA Delivery
An Approach to Cancer Therapy

2. Gene Delivery Slide 2 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Definitions
 Gene therapy; refers to the transmission of nucleic acid (DNA
and RNA) encoding a therapeutic gene of interest into the
targeted cells or organs with consequent expression of the
transgene.
 Gene Delivery: is the process of introducing foreign genetic
material into host cells.
In other words, Gene Delivery is a key step to gene therapy.
 A Gene Delivery System (GDS) is a special purpose conjugate
that consists of carry-over material and nucleic acids (payload).

3. Gene Delivery Slide 3 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Gene Therapy: Approaches
 Several Approaches may be used for correcting faulty
genes:
– A normal gene may be inserted into the genome to compensate
for nonfunctional gene. (the most common approach)
– An abnormal gene is replaced for a normal gene
– The abnormal gene could be repaired through selective reverse
mutation.
– The regulation of a gene expression could be altered.

4. Gene Delivery Slide 4 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Barriers to Gene Delivery
 In order to deliver genes into cell nucleus, several barriers need to
be overcome:
 Extracellular barriers:
– Nucleases
– Plasma Proteins (aggregation)
– Immune system
– Glomerular filtration
 Cellular barriers:
– Cell Membrane
– Endolysosomal entrapment
– Cytosolic Sequestration
– Nuclear Exclusion

5. Gene Delivery Slide 5 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery: Off-target Effect
 siRNAs are capable of reducing expression of nontarget genes due
to interaction of the siRNA guide strand with a partially
complementary site on an “off-target” mRNA.
 Careful selection of siRNA sequences to avoid off-target effects is
an important issue.

6. Gene Delivery Slide 6 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery: Immune stimulation
 Innate immune activation is a significant undesirable side effect
due to the toxicities associated with excessive cytokine release.
 The inflammatory response is mediated by toll-like receptors (TLRs)
which are located in endosome compartments, and recognize
unmethylated CpG DNA as well as various moieties in double-
stranded RNA or their degraded products.
 TLR-mediated recognition and concomitant immune stimulation
can be inhibited by chemical modifications, such as introduction
of 2′-O-methyl (2′OMe)-modified nucleotides.

7. Gene Delivery Slide 7 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Serum inactivation and enzyme degradation
 Naked genetic material are rapidly degraded by nuclease in the
serum.
 2′-hydroxyl group of the ribose ring is not necessary for gene
silencing by siRNAs, common modifications are 2′-fluoro, 2′-O-
methyl, and 2′-amine conjugations.
 Naked genetic material lack specific tumor targeting and would be
quickly excreted by the kidney upon systemic administration.

8. Gene Delivery Slide 8 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
RES Recognition
 In order to condense negatively charged nucleic acids into
delivery vehicles, most vehicles are cationic.
 The positive charge aids cellular uptake but also promotes
nonspecific interactions with non target cells and extracellular
components such as serum proteins and extracellular matrix.
 Binding of proteins is the primary mechanism for RES recognition.
 The most common way to decrease nonspecific interactions is
to shield the nanoparticle surface with hydrophilic, uncharged
polymers such as polyethylene glycol (PEG)

9. Gene Delivery Slide 9 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Entrance Into Cells
 Delivery systems that are not degraded, phagocytosed or cleared by the
kidney must leave the bloodstream by traversing the endothelium to reach
other tissues. This occurs most readily in tissues whose endothelia are
discontinuous (fenestrated), such as the liver and many solid tumours.
 Most siRNA delivery systems undergo cellular internalization through
endocytosis.
 Various delivery systems aim to improve the rate of cellular uptake by
incorporating targeting ligands that bind specifically to receptors on target
cells to induce receptor-mediated endocytosis.
 Other systems use cell-penetrating peptides that can induce cell uptake
through endocytosis or non-endocytic mechanisms.

10. Gene Delivery Slide 10 of 100
December 2013
Tehran University of
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School of Pharmacy
Challenges to Gene Delivery:
Entrance Into Cells
 Commonly used targeting ligands include
– Aptamers
– Cell penetrating peptides
– Antibodies
– Peptides or proteins
– Small molecule ligands

11. Gene Delivery Slide 11 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Endosome Escape
 The delivery of nonviral gene vehicles almost invariably involves endocytosis,
and escaping from endosomes before they traffic into lysosomes is an essential
step to avoid enzymatic degradation.
 Cationic lipid complexes can bind to anionic lipids on the endosome
membrane and form neutral ion pairs. These ion-pairs destabilize the endosome
membrane and promote de-assembly of the lipoplex
 Polymers and peptides with high buffer capacity between pH 7.2 and 5.0, such
as PEI, or peptides containing the cationic amino group lysine, arginine, and
histidine, could buffer the endosome. This would cause more protons to
enter into the endosomes, followed by chloride ions, leading to increased
osmotic pressure and endosome rupture. (“proton sponge effect”)
 Stimuli other than pH have been used to destabilize endosome membranes.
Lipid or polymer derivatives which are sensitive to sulfhydryl reduction and
enzymatic cleavage have been used to construct nonviral gene vectors.

12. Gene Delivery Slide 12 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Endosome Escape

13. Gene Delivery Slide 13 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Nuclear Entry
 The final target destination of antisense oligonucleotides,
siRNA/miRNA, and mRNA, is the cytoplasm, whereas pDNA must
be transported into the nucleus for gene expression.
 Nuclear transport generally occurs through nuclear pore
complexes(NPCs), however, nucleic acid condensates are
impermeable through nuclear pore complexes due to their large size.
 In dividing cells, the nuclear envelope disassembles during
mitosis; pDNA transfection can only occur at this stage
 For nondividing cells, the mechanisms of DNA nuclear transport are
of critical importance.

14. Gene Delivery Slide 14 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Nuclear Pore Complex

15. Gene Delivery Slide 15 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
 The NPC is freely permeable to small molecules, metabolites and
ions, but acts as a highly efficient molecular sieve for macromolecules
 Transport of almost all macromolecules into and out of the nucleus is
achieved requires the assistance of soluble nuclear transport
factors (NTFs) and transport signals.
 The NTFs specifically bind transport signals found on their cognate
substrates and translocate them through the NPC channel.
 The best-studied transport signals are found on nuclear protein
cargoes. Such signals consist of short amino acid sequences called
nuclear localization sequences (NlSs; for import) or nuclear export
sequences (NESs; for export)
Nuclear Pore Complex

16. Gene Delivery Slide 16 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Gene Delivery:
Nuclear Entry
 To facilitate nuclear targeting, many nuclear localization signal
(NLS) peptides have been studied to allow DNA nuclear entry
through nuclear pore complexes by active transport.
 NLSs are short clusters of amino acids that can bind to
cytoplasmic receptors known as importins. NLS peptides can
bind to DNA either through noncovalent electrostatic interaction
or by covalent attachment.
 The most well-known and popularly used NLS is from the large
tumor antigen of simian virus 40 (SV40).
 Some DNA sequences themselves have nuclear import activity
based on their ability to bind to cell-specific transcription factors

17. Gene Delivery Slide 17 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Gene Delivery Systems
 The most important and most difficult challenge in gene therapy is
the issue of delivery.
 Not only must the therapy evade the RES as it circulates after
systemic administration, but it must also cross several barriers
before it arrives in the cytoplasm or nucleus of its target cells.
 So there is a need to develop a safe and efficient gene transfection
therapy system.
 Carry-over material of a GDS can pass through the cell membrane,
and in some instances, enter the nucleus.
 Carry-over material are called vectors.

18. Gene Delivery Slide 18 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Vectors
 A vector can be described as a system fulfilling several
functions, including
– Enabling delivery of genes into the target cells and their nucleus
– Providing protection from gene degradation
– Ensuring gene transcription in the cell.
 Gene therapy vectors should not only be targeted and safe, but also
protected from degradation, sequestration, or immune attack.
 However, it has to be inexpensive and easy to produce and purify in
large amounts and at high concentrations.

19. Gene Delivery Slide 19 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Categorization
 Gene delivery systems can be classified into two categories based
on the origin of the gene carrier:
– Viral gene delivery systems use recombinant viruses as carriers
– Non-viral gene delivery system
• Physical (carrier-free gene delivery)
• Chemical (synthetic vector-based gene delivery
 Each vector has its own advantages and disadvantages.
 None of these types of vectors has been found to be ideal for both
safe and efficient gene transfer and stable and sufficient gene
expression.

20. Gene Delivery Slide 20 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Viral Vector Delivery Systems
 Viral techniques use various classes of viruses for gene delivery.
 Viruses introduce their genome into the cells with high efficiency.
 However, there are limitations, including a strong immune response
triggered by the expression of viral genes, oncogenic insertions into
the genome, and unstable maintenance of viruses in the host cell.
 Gene therapy vectors are being developed by genetic modification
of retroviruses, adenoviruses, poxviruses, parvoviruses (adeno-
associated viruses), herpesviruses, and others.

21. Gene Delivery Slide 21 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Viral Vector Replication
 These vectors are engineered by deleting the essential genes
which allow replication, assembling, or infection.
 But on the other hand, vectors need to be produced in large
amounts of virus particles.
 Specialized cell lines called “packaging cell lines” (PCLs)
engineered to replace a function of a deleted viral gene.
Removed virus genes are inserted into the genome of the
packaging cells and can be expressed there.
 The process by which the function of the deleted viral gene is
supplied by a protein encoded in the PCL genome is called
“complementation”

22. Gene Delivery Slide 22 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Viral Vectors

23. Gene Delivery Slide 23 of 100
December 2013
Tehran University of
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Non-Viral Delivery Systems
 Non-viral techniques of gene transfer represent a simple and, more
importantly, safer alternative to viral vectors.
 Because of their relatively simple quantitative production and low
immunogenicity, these vectors are attractive tools in gene therapy.
 Ongoing studies and the development of new vectors that show
transfection efficiency just like that of viral vectors point towards
their great potential.
 The main advantage is a virtually unlimited clone capacity, low
toxicity and immunogenicity of non-viral vectors, and the ability of
repeated application.

24. Gene Delivery Slide 24 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges to Non-viral Gene Delivery
 Viruses are naturally occurring being which are specialized for
transfection.
 So, they inherently can pass the barriers to gene delivery and
transfer their genetic material into host
 For non-viral delivery systems, these abilities should be designed in
the vector.

25. Gene Delivery Slide 25 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Non-viral Gene Delivery Systems
 Non-viral gene delivery system can be categorized to:
– Physical (carrier-free gene delivery)
• Naked DNA
• Direct Injection
• Electroporation
• Gene-Gun
– Chemical (synthetic vector-based gene delivery)%
• Lipoplex Vectors
• Polyplexes
• Dendrimeric Vectors
• Polypeptide Vectors
• Inorganic Nanoparticles

26. Gene Delivery Slide 26 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Non-Viral Delivery Systems:
Lipoplexes and Polyplexes
 Synthetic vectors improving the admission of DNA into the cell and
protecting it from undesirable degradation were created.
 The most used are derived from lipids or synthetic polymers.
 Plasmid DNA can be covered by lipids into organized structures
such as liposomes or micelles. This complex of DNA with lipids is
called a lipoplex.
 Vectors based on a complex of polymers with DNA are called
polyplexes.

27. Gene Delivery Slide 27 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Lipoplexes
 Lipoplexes can be divided into two types:
– Anionic and neutral liposomes: are characterized by safety,
compatibility with body fluids, and the possibility of tissue-
specific gene transfer, but the level of transduced cell
expression is relatively low.
– Cationic and Ionizable liposomes: naturally create complexes
with negatively charged DNA, Their positive charge, allows
interactions with the negatively charged cell membrane
 Targeted transfection can be gained, to some extent, by the
addition of tissue-specific target ligand.

28. Gene Delivery Slide 28 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polyplexes
 Most of Polyplexes consist of cationic polymers and their
production is regulated by ionic interactions.
 In contrast to Lipoplexes, some Polyplexes (poly lysin) are
not able to release intracellular DNA into the cytoplasm
 Polymers such as polyethylenimine have a mechanism of
endosome disruption and there is thus no need for
transfection with endosome-lytic agents.

29. Gene Delivery Slide 29 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polyplexes: Poly(L-Lysine) or PLL
 Polylysine has an exceptional capacity to condense DNA
 At physiological pH, all primary-amino groups of PLL are
protonated, yielding a structure with no buffering capacity to aid in
endosomal escape.
 Polylysine structures with molecular weights>3000 Da can
effectively condense DNA, indicating the significance of primary
amine number for complex formation.
 But these heavier PLLs exhibit relatively high cytotoxicity.
 This toxicity has been reduced with the incorporation of imidazole
group into the PLL chain, or through the use of dendritic PLLs.

30. Gene Delivery Slide 30 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polyplexes: Poly(L-Lysine) or PLL
 High MWt. PLL/DNA complexes also have a tendency to aggregate
and precipitate depending on the ionic strength of the solution.
 One method used to overcome the formation of insoluble
precipitates is to form block copolymers of PLL with PEG.
 To overcome nonspecific cell targeting, various research groups
have derivatized PLL with targeting moieties.

31. Gene Delivery Slide 31 of 100
December 2013
Tehran University of
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School of Pharmacy
Polyplexes: Polyethylenimine (PEI)
 Polyethylenimine is often considered the gold standard of gene
transfection.
 Polyethylenimine exists as both a branched and linear structure.
 The transfection efficiency of PEI is due, at least in part, to the
“proton sponge” nature of it.
 At a physiological pH, approximately 80% of the amines remain
unprotonated compared to less than 50% unprotonated nitrogens
at a pH of 5.
 This buffering capacity allows PEI polyplexes to avoid lysosomal
trafficking and degredation once inside the cell.

32. Gene Delivery Slide 32 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polyplexes: Polymethacrylate
 Due to its inherent cationic charge, poly[2-(dimethylamino)ethyl
methacrylate] (PDMAEMA) offers significance as a gene transfer agent.
 The successful in vitro transfection efficiency of PDMAEMA
polyplexes is attributed to the ability of the polymer to destabilize
endosomes as well as to dissociate easily from the plasmid once
delivered into the cytosol.
 The mechanism of gene transfer for methacrylate polyplexes has
been shown to proceed by both clathrin- and caveolae-dependent
pathways.
 Caveolae-dependent uptake appears to be vital for effective gene
transfer of PDMAEMA polyplex.

33. Gene Delivery Slide 33 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Carbohydrate Based Polymers: β-cyclodextrins
 To overcome the cytotoxicity, incorporating β-cyclodextrin into cationic
polymers has been studied.
 The length of the alkyl chain(n) between cyclodextrin monomer units
affects polyplex cytotoxicity:
– Cytotoxicity generally decreases with longer chain lengths (decreased
charge density)
– With alkyl chain lengths (n) ranging from 4 to 10, cytotoxicity was lowest
– Transfection efficiency was highest with 6, 7, or 8 methylene units.
– The high toxicity/low transfection efficiency of the polymer with 10 methylene
units was attributed to lower solubility.
 Like most other cationic vector systems, in vivo use of β-cyclodextrin is
hindered by aggregation at high ionic strengths. While PEGylation can
generally reduce the formation of aggregates

34. Gene Delivery Slide 34 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Carbohydrate Based Polymers: Chitosan
 The biodegradability, biocompatibility, and cationic potential of
chitosan has helped it become one of the most prominent, naturally
derived nonviral vectors for gene transfer.
 Chitosan is produced by deacetylation of chitin to form a polymer
composed of D-glucosamine and N-acetyl D-glucosamine subunits
linked by β(1,4) glycosidic bonds.
 Molecular weight of chitosan polymers can strongly influence gene
transfer efficiency.
 Regardless of the increased polyplex size, high molecular weight
chitosan forms more stable complexes with DNA due to a chain
entanglement effect.

35. Gene Delivery Slide 35 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Carbohydrate Based Polymers: Chitosan
 In addition to molecular weight, several other factors have been
shown to affect the transfection efficiency of chitosan polyplexes,
including the N/P ratio, pH and the degree of deacetylation.
 Optimal transfection efficiency of chitosan polyplexes can be
achieved between pH 6.8 and 7.0.
 Above pH 7.5, DNA was shown to dissociate from the complex.
 Below pH 6.5, cellular uptake was significant but transfection
efficiency was low, possibly due to hindered endosomal release.

36. Gene Delivery Slide 36 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Dendrimer Based Vectors: PAMAM
 Polyamidoamine Dendrimers (PAMAM):
– Due to ease of synthesis and commercial availability, they have become
the most utilized dendrimer-based vectors for gene transfer.
– High generation PAMAM dendrimers (G5-G7) induce lipid mixing and
leakage from phospholipid vesicles. Which was attributed to the ability
of the spherical PAMAM structure to bend the anionic membrane
through electrostatic forces and induce packing stresses, leading to lipid
mixing.
– Following cellular uptake, endosomal chloride accumulation increased
significantly and the pH also increased, indicating the occurrence of
endosomal swelling/lysis. (proton sponge effect)
– Various alterations to the basic PAMAM dendrimer structure have been
investigated in an effort to improve transfection efficiency or
PAMAM/DNA complex formation.

37. Gene Delivery Slide 37 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polypeptide Vectors
 Peptide-oligonucleotide conjugates offer a strategy of delivering
genetic material into cells with high efficiencies and cell-specificity.
 These amino acid sequences, called cell-penetrating peptides(CPPs),
are generally divided into two classes:
– lysine-rich peptides, such as the amphipathic MPG peptide and transportan
– arginine-rich peptides, such as the homeodomain of antennapedia (Antp) and
trans-activating transcriptional activators (TAT)
 Generally the peptide is covalently linked to the oligonucleotide
construct rather than complexed via electrostatic interactions.
(Complexation of peptides and siRNA can be either electrostatic or covalent).

38. Gene Delivery Slide 38 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polypeptide Vectors: TAT-Based Peptides
 TAT protein is an 86-102 amino acid sequence organized into three
domains:
– cationic regions involved in controlling the rate of gene expression
– cysteine-rich regions involved in DNA binding, and
– basic amino acid regions involved in promoting the crossing of the membrane.
 The cellular uptake of free Tat-peptides has been shown to proceed
by an energy-independent pathway, but the transfection of Tat-DNA
complexes may proceeds by endocytosis.
 As an example: By covalently attaching the Tat-peptide with anti-
MDR antisense oligonucleotide, it was shown that in vitro P-
glycoprotein expression could be significantly inhibited

39. Gene Delivery Slide 39 of 100
December 2013
Tehran University of
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School of Pharmacy
Polypeptide Vectors: Antennapedia Homeodomain Peptide
 Antennapedia homeodomain is a 60-amino acid polypeptide corresponding to the
Drosophilia melanogaster antennapedia homeobox sequence.
 While the third alpha-helix of the pAntp is involved in promoting translocation, the
60-amino acid structure could be reduced to a 16-mer peptide.
 Cellular uptake of pAntp proceeds by a nonendocytic pathway.(Even at 4ºC)
 The replacement of several amino acids with proline disrupted the alpha-helical
structure of pAntp but did not hinder cellular uptake, suggesting that the alpha-
helical conformation is not required.

40. Gene Delivery Slide 40 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Polypeptide Vectors: MPG peptide
 The MPG peptide is a synthetic compound containing a
hydrophobic N-terminal region derived from the fusion sequence of
HIV and a hydrophilic region derived from the NLS of the SV40
large T antigen
 Complexation of MPG with oligonucleotides proceeds via
electrostatic interaction between the basic residues of the NLS
region and the phosphonate backbone of the oligonucleotide.
 When MPG peptide is complexed with oligonucleotides, the
hydrophobic region partially folds into a β-sheet structure, which
promotes cellular uptake by inserting into the plasma membrane
and forming a transmembrane pore-like structure.

41. Gene Delivery Slide 41 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Mechanisms of membrane translocation
 Theories of CPP translocation can be classified into three main
entry mechanisms:
 Direct penetration in the membrane
– Most likely involved a direct electrostatic interaction with negatively
charged phospholipids.
– interactions between cell-penetrating peptides and the phosphate groups on both
sides of the lipid bilayer, the insertion of charged side-chains that nucleate the
formation of a transient pore, followed by the translocation of cell-penetrating
peptides by diffusing on the pore surface.
 Endocytosis-mediated entry
 Formation of a transitory structure
– Formation of the inverted micelles.
– Transmembrane structures

42. Gene Delivery Slide 42 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Inorganic Nanoparticles
 The application of inorganic nanoparticles in gene delivery is an
emerging field, too, because they can be prepared and surface-
functionalized in many different ways. Examples of such systems are:
– Metallic nanoparticles (Gold nanoparticles are inert and are easily functionalized)
– Iron oxide (superparamagnetic, cytotoxic, used in polymer-coated form)
– calcium phosphate, magnesium phosphate, manganese phosphate
– carbon nanotubes (are surface functionalized and used)
– quantum dots (employed for tracking of delivery systems)

43. Gene Delivery Slide 43 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Nanoparticles: Iron Oxide Nanoparticles
 Iron oxide nanoparticles (IONPs), superparamagnetic material
sized between approximately 1 and 100 nm, have a long history of
investigation.
 In order to produce IONPs that are highly efficient for gene delivery,
the IONPs should have an enhanced cationic surface with cationic
polymers such as PEI, PLL, and chitosan.
 PEI-IONPs are limited for in vivo applications due to cellular
toxicity.
 In order to overcome cellular toxicity, various polymers have been
utilized to coat or conjugate to the IONPs. (PEG for example)

44. Gene Delivery Slide 44 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Nanoparticles: Carbon nanotubes
 Carbon nanotubes can be used as a GDS when they are
chemically modified.
 They can be either covalently functionalized by oxidation and
subsequent 1, 3-dipolar cyclo-addition reaction or non-covalently
functionalized with hydrophobic or π-π stacking between the CNT
and another non-polar ring such as the backbone of DNA.
 In vitro and in vivo studies have suggested that PEG-modified
CNTs have favorable pharmacokinetic and toxicology profiles.

45. Gene Delivery Slide 45 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Nanoparticles: Silica nanoparticles
 Silica nanoparticles (SiNPs) have been used as drug and gene
delivery agents because they can be easily modified.
 SiNPs need to be modified with an anchoring group and charge
transfer functional group to allow for DNA binding by electrostatic
interactions for efficient cellular delivery.
 The regular arrangement of pores or hollow cavities in the silica
nanoparticles easily accommodates siRNA molecules.
 Silica materials are usually toxic, but their toxicity may be reduced
through surface modifications. For example, surface-coating with
PEI makes positively charged SiNPs which can pass through the
cell membrane more easily and have significantly reduced toxicity.

46. Gene Delivery Slide 46 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Nanoparticles: Gold nanoparticles
 Gold nanoparticles (AuNPs) consist of colloidal gold suspended in
liquids, sized from 1 to 150 nm.
 Gold nanoparticles have emerged as an attractive and widely used
nanomaterial for GDSs because they are inert and essentially
nontoxic to cells.
 Furthermore, AuNPs can be easily functionalized by anchoring thiol
linkers. Conjugate materials used for facilitating cellular uptake
include peptides, proteins, antibodies, oligosaccharides, and
nucleic acids.

47. Gene Delivery Slide 47 of 100
December 2013
Tehran University of
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Nanoparticles: Gold nanoparticles
 Antisense oligodeoxynucleotide (ASODN)-modified gold
nanoparticles have higher affinity for complementary nucleic acids
than their unmodified oligonucleotide counterparts.
 Furthermore, AuNP-ASODNs are less susceptible to degradation
by nuclease activity, exhibit greater than 99% cellular uptake, and
are less toxic to the cells under the studied conditions.

48. Gene Delivery Slide 48 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges in Non-viral GDS
 Non-viral GDSs lack mechanisms for integration into the host
chromosome.
 So, the expression of delivered genes will be transient, because it
gradually becomes inactivated.
 One the inactivation mechanisms is so-called “gene dilution”. This
process takes place mainly in dividing cells with a plasmid vector
that is not integrated and the number of copies of
extrachromosomal DNA reduces with each cell cycle.

49. Gene Delivery Slide 49 of 100
December 2013
Tehran University of
Medical Sciences
School of Pharmacy
Challenges in Non-viral GDS
 For this reason, combination of DNA transposon-based vectors is
required for gene delivery.
 A transposon is a DNA sequence that can change its position
within the genome through a direct cut-and-paste mechanism.
 A simple transposon is organized by terminal inverted repeats
embracing a gene encoding transposase, an enzyme required for
its transposition.
 Engineered DNA transposons have the
desired features of naked DNA and plasmids
as well as the ability to insert transgenes into
host chromosomes for long-term transgene
expression.

50. Gene Delivery Slide 50 of 100
December 2013
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Transposition

51. Gene Delivery Slide 51 of 100
December 2013
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RNA Interference
 RNA interference (RNAi) gained international attention in 1998
when Fire, Mello and colleagues discovered the ability of double-
stranded RNA to silence gene expression in the nematode worm
Caenorhabditis elegans.
 Three years later, Tuschl et. al. published their experiment
demonstrating that synthetic siRNA could achieve sequence-
specific gene knockdown in a mammalian cell line.
 The first successful use of siRNA for gene silencing in mice was
achieved for a hepatitis C target shortly thereafter

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RNA Interference
 There are numerous classes of small non-coding RNA in eu. Cells
involved in RNA splicing, editing, and modification. (catalytic RNAs)
 Very small RNAs or micro RNAs (miRNA) are gen expression
regulators found in most eu. cells.
 Human genome hast an estimated 1000 genes
that code for miRNAs that participate in RNA
interference(RNAi) (half from coding genes and
half from ncRNAs or even pseudogenes)
 This is a general mechanism to repress gene
expression, usually but not always, at the
translation level.

53. Gene Delivery Slide 53 of 100
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RNA Interference
 These miRNAs go by a number of names and are sometimes
called short temporal RNAs (stRNA) because of their role in
development.
 Piwi-associated RNAs(piRNA) are found in germ cells.
 Small interfering RNAs(siRNA) are typically produced during a viral
infection.
 Both can be used to control the expression of transposable
elements.

54. Gene Delivery Slide 54 of 100
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RNA Interference
 RNAi is triggered by the presence of long pieces of double-stranded
RNA, which are cleaved into the fragments known as siRNA (21–23
nucleotides long) by the enzyme Dicer.
 In practice, siRNA can be synthetically produced and then directly
introduced into the cell, thus circumventing Dicer mechanics.

55. Gene Delivery Slide 55 of 100
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56. Gene Delivery Slide 56 of 100
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Tehran University of
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RNA Interference
 RNAi is triggered by the presence of long pieces of double-stranded
RNA, which are cleaved into the fragments known as siRNA (21–23
nucleotides long) by the enzyme Dicer.
 In practice, siRNA can be synthetically produced and then directly
introduced into the cell, thus circumventing Dicer mechanics.
 Once siRNA is present in the cytoplasm of the cell, it is incorporated
into a protein complex called the RNA induced silencing
complex(RISC) composed of argonauts(ago) protein family.
 RISC has endonuclease activity that cleaves the passenger strand
the one which will not be used in the duplex siRNA or miRNA.

58. Gene Delivery Slide 58 of 100
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RNA Interference
 The degree of base pairing and the sequence of the ends
 The RISC complex is now in a position to use the mature siRNA to
guide it to its target mRNA.
 siRNA searches mRNAs for small regions of homology usually
found in AU-reach region in 3’-UTR
 Two primary mechanisms used to control mRNA expression:
– Degredation of the mRNA (common in plant cells)
– Inhibition of translation (common in animals)
 The choice is primarily determined by the degree of base-pairing
between the siRNA and mRNA. The higher base paring the more
likely that the target mRNA will be degraded.

60. Gene Delivery Slide 60 of 100
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RNA Interference
 The activated RISC complex can destroy additional mRNA
targets, which further propagates gene silencing. This extra
potency ensures a therapeutic effect for 3–7 days in rapidly
dividing cells, and for several weeks in non-dividing cells.
 Theoretically, when using appropriately designed siRNA, the
RNAi machinery can be exploited to silence nearly any gene in
the body, giving it a broader therapeutic potential than typical
small-molecule drugs.
 In order for these advances to be implemented in a clinical
setting, safe and effective delivery systems must be developed.

61. Gene Delivery Slide 61 of 100
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Delivery Systems
 Naked, chemically modified siRNA has shown efficacy in certain
physiological settings such as the brain and the lung.
 But most tissues in the body require an additional delivery system to
facilitate transfection.(enzymes, large size(~13 kDa) too negatively charged)
 The issue of effective and non-toxic delivery is a key challenge and
serves as the most significant barrier between siRNA technology
and its therapeutic application.

62. Gene Delivery Slide 62 of 100
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Delivery Systems
 localized siRNA delivery:
– Application of siRNA therapy directly to the target tissue
– Several tissues are amenable to topical or localized therapy,
including the eye, skin, mucus membranes, and local tumors.
– well-suited for the treatment of lung diseases and infections.
The direct instillation of siRNA into the lung through intranasal
or intratracheal routes.

63. Gene Delivery Slide 63 of 100
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siRNA Modification
 Chemical modifications can be introduced
into the siRNA molecule to evade immune
defense and nucleases in vivo.
 Incorporation of 2′-O-methyl or 2′-Fluoro
or 2-methoxyethyl modifications into
the sugar structure of selected nucleotides
within both the sense and antisense strands.
 Replacement of the phosphodiester(PO4)
group with phosphothioate(PS) at the 3'-end
 The locked nucleic acid substitution
containing a methylene linkage between the 2'- and 4'-positions

64. Gene Delivery Slide 64 of 100
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Polymer Vectors
 siRNA differs from pDNA in Mwt, charge ratio, stability and action.
However, both are nucleic acids.
 Polymer-mediated DNA delivery systems would provide a lot of
knowledge for the development of polymer-based siRNAs.
 Cationic polymers usually form a complex with negatively charged
siRNA upon simple mixing.
 Categories:
– Synthetic: PEI, PLL, cyclodextrin-based polycations
– Natural: Chitosan, atelocollagen and polypeptides

65. Gene Delivery Slide 65 of 100
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Cyclodextrin Polymer Vectors
 Cyclodextrin polymers are polycationic oligomers (n ≈ 5)
synthesized by a step-growth polymerization between diamine-
bearing cyclodextrin monomers and dimethyl suberimidate, yielding
oligomers with amidine functional groups.
 The strong basicity of these amidine groups mediates efficient
condensation of nucleic acids.
 End-capping of the polymer termini with imidazole functional group
can aid endosomal escape.
 Both adamantane–PEG (AD–PEG) and adamantane–PEG–
transferrin (AD–PEG–Tf) were incorporated to improve particle
properties.

66. Gene Delivery Slide 66 of 100
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Cyclodextrin Polymer Vectors

67. Gene Delivery Slide 67 of 100
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Cyclodextrin Polymer Vectors

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Lipid Vectors
 Liposomes studied as delivery vectors for DNA-based drugs
because of their ability both to protect entrapped oligonucleotides
from nucleases and renal clearance, and to promote cellular uptake
and endosomal escape.
 Cationic and ionizable lipids:
– Improve the entrapment of the negatively charged siRNA, increase cellular
uptake and aid endosomal escape
– Several studies have determined that cationic lipids, are less efficacious and
more toxic than ionizable lipids, whose charge is dependent on the pH
– Thus, recent work has focused on the development of new ionizable lipids.
– The composition of these lipids is generally divided into three parts:
an amine head group, a linker group and hydrophobic tails

69. Gene Delivery Slide 69 of 100
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Lipid Vectors: Ionizable Lipids
 To minimize toxicity without sacrificing efficacy, the pKa of an
ionizable lipid should be low enough for it to remain unprotonated
during circulation but high enough for it to become protonated in
either the early or late endosome. (5.4-7.6)
 Lipid transition temperature refers to the temperature at which lipid
membranes shift from the more stable lamellar phase to the less
stable hexagonal phase.
 This transition promotes destabilization of the endosomal membrane
and release of siRNA

70. Gene Delivery Slide 70 of 100
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Lipid Vectors: Ionizable Lipids
 Lipids with lower transition
temperatures, which more readily
shift from lamellar to hexagonal to
promote endosomal release, have
small polar head groups and large
unsaturated hydrophobic tails. Lipids
with large polar head groups and
fully saturated hydrophobic tails are
more likely to adopt the stable
lamellar phase.
Successful lipid formulations are
engineered to remain in the lamellar
phase during circulation and to
transition to the hexagonal phase
within endosomal compartments.

71. Gene Delivery Slide 71 of 100
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Lipid Vectors
 Shielding lipids:
 Lipid-anchored PEG is a common component in liposomes.
 PEG groups serve many purposes: they prevent aggregation,
increase circulation time and reduce uptake by unintended targets
such as RBCs and macrophages.
 Shielding lipids can reduce cellular uptake and efficacy.
 After endocytosis, PEG can sterically and electrostatically block the
interaction between the liposome and the endosomal membrane,
hindering membrane fusion and preventing endosomal release.
 One strategy for improving the efficacy
of PEGylated nanoparticles involves
incorporation of acid-sensitive bonds
connecting PEG to the liposome.

72. Gene Delivery Slide 72 of 100
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Lipid Vectors: Shielding Lipids
 Another method to reduce the negative effects of shielding involves
the use of a pH-sensitive modified PEG that binds to liposomes
through ionic interactions.
 The liposomal core consists of 1,2-dioleoyl-sn-glycero-3-
phosphatidylethanolamine (DOPE) as well as 2-hydroxyethyl
methacrylate(HEMA)–lysine-modified cholesterol
 PEG is covalently modified with HEMA–histidine–methacrylic acid.
 At neutral pH the PEG copolymer has a net negative charge
whereas the liposomal core has a net positive charge. In the
endosome, imidazole and methacrylic acid residues become
protonated, and the net charge of the PEG becomes positive.

73. Gene Delivery Slide 73 of 100
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Lipid Vectors
 Cholesterol:
 Many liposomal formulations include cholesterol, which can
associate with lipid bilayers.
 Increase in the cholesterol content lowers the transition temperature
of liposomal membranes containing conical-shaped lipids.

74. Gene Delivery Slide 74 of 100
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Lipid Vectors
 Targeting ligands
 To improve the biodistribution of liposomes, many formulations use
endogenous or exogenous targeting ligands.
– Endogenous targeting ligand, often a serum protein, binds to the liposome
during circulation and directs it to the ligand’s natural target.
• The lipoprotein ApoE has been used as an endogenous targeting ligand by DLin–KC2–
DMA-based ionizable liposomes
• Retinol binding protein (RBP) is also used an endogenous targeting ligand. This serum
protein binds vitamin A and transports it to cells expressing the RBP receptor, including
hepatic stellate and pancreatic stellate cells.
– Exogenous targeting ligands are added to liposomal formulations before
injection to bind desired surface proteins on target cells.
• folate, which has been used to target delivery to rapidly dividing cancer cells is an
example of exogenous ligands.

75. Gene Delivery Slide 75 of 100
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Lipid Vectors: SNALPs
 For in vivo siRNA delivery, stable nucleic acid–lipid particles
(SNALPs) have been formulated.
 A SNALP consists of a lipid bilayer containing a mixture of cationic
and fusogenic lipids that enables the cellular uptake and
endosomal release of siRNA.
 The surfaces of SNALPs were coated with
a polyethylene glycol–lipid conjugate that
provides a neutral, hydrophilic, exterior and
stabilizes the particle during formulation

76. Gene Delivery Slide 76 of 100
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Conjugate delivery systems
 Directly conjugating delivery material to the siRNA cargo.
 The first conjugate delivery systems to show efficacy in vivo
consisted of siRNA conjugated to cholesterol and other lipophilic
molecules.
 Other conjugate delivery systems have been developed by
attaching siRNA to polymers, peptides, antibodies, aptamers and
small molecules.
– Dynamic PolyConjugates
– GalNAc–siRNA

77. Gene Delivery Slide 77 of 100
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Conjugate Delivery Systems: Dynamic Polyconjugates
 siRNA–polymer conjugate delivery systems designed to respond to intracellular
environments.
 The siRNA cargo is attached to a membrane-disrupting polymer by a hydrolysable
disulphide linker, cleaved in the reducing environment of the cytosol, releasing the
siRNA from the delivery polymer.
 The activity of the polymer is masked by PEG side chains while the system is in
circulation. The PEG is designed to be shed in the acidic environment inside the
endosome.
 To induce uptake by target cells through receptor-mediated endocytosis, ligands
are incorporated. GalNAc ligands, which bind to the asialoglycoprotein receptor
(ASGPR) on hepatocytes.
 the siRNA itself is chemically modified to improve nuclease stability and to reduce
off-target effects.

78. Gene Delivery Slide 78 of 100
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Conjugate Delivery Systems: Dynamic Polyconjugates

79. Gene Delivery Slide 79 of 100
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Conjugate Delivery Systems: Triantennary GalNAc–siRNA
 In this system the 3ʹ terminus of the siRNA sense strand is
attached to three GalNAc molecules by means of a triantennary
spacer.
 Multivalency of the sugar ligand greatly improves cell uptake, and
spacing of the sugar moieties also plays a role. In a study of
triantennary galactose ligands, binding affinity increased with
spacer length over a range of 4–20 Å

80. Gene Delivery Slide 80 of 100
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Oligonucleotide Vectors
 The construction of three-dimensional nanoparticles of defined
composition from nucleic acids has generated interest:
– Molecularly identical nanoparticles with strictly defined characteristics
 Oligonucleotide nanoparticles (ONPs) were composed of
complementary DNA fragments designed to hybridize into
predefined three-dimensional structures.
 constructing DNA tetrahedra was adapted by incorporating single-
stranded overhangs on each edge.
 Short interfering RNAs were modified by extension of the 3ʹ sense
strands with DNA overhangs.

81. Gene Delivery Slide 81 of 100
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Oligonucleotide Vectors

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Oligonucleotide Vectors
 Oligonucleotide nanoparticles modified with folate ligands were
used to study the minimum number of targeting ligands required
 These questions are difficult or impossible to address using many
other nanoparticle systems.

83. Gene Delivery Slide 83 of 100
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Cancer
 Worldwide, between 100 and 350 of each 100,000 people die of cancer
each year.
 Genetic control systems regulate the balance between cell birth and death
in response to growth signals, growth-inhibiting signals, and death signals.
 The losses of cellular regulation that give rise to most or all cases of cancer
are due to genetic damage
 Mutations in two broad classes of genes have been implicated in the onset
of cancer: proto-oncogenes and tumorsuppressor genes.
 Many of the genes in both classes encode proteins that help regulate cell
birth (entry into and progression through the cell cycle) or cell death by
apoptosis; others encode proteins that participate in repairing
damaged DNA

84. Gene Delivery Slide 84 of 100
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Cancer
 Seven types of proteins that
participate in controlling cell growth
and proliferation.
– Extracellular signaling molecules (I),
– Signal receptors (II),
– Signal-transduction proteins (III),
– Transcription factors (IV)
– Apoptotic proteins (V)
– Cell cycle control proteins (VI)
– DNA-repair proteins (VII)

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86. Gene Delivery Slide 86 of 100
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Tumor Targeting
 Can be categorized to Passive Targeting and Active Targeting
 Passive Targeting:
Enhanced Permeability and Retention (EPR) effect
– For most tumors, nanoparticles with a mean size around 100 nm are
attractive for tumor targeting.
– Particles larger than 400 nm can not easily enter the capillary gaps in the
tumor vasculature, whereas particles smaller than 70 nm are able to
access the parenchymal cells in the liver.

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Tumor Targeting
 Active Targeting:
 Grafting markers or ligands at the surface of the delivery system for
receptors only expressed or at least overexpressed in tumors.
 Tumor homing peptides are able to react with different receptors:
– Integrin receptors with RGD peptide (ArgeGlyeAsp)
Alpha V integrin proteins overexpressed on tumoral vessels
– AsneGlyeArg (NGR) motif interact with aminopeptidase N receptor (CD13)
The expression of this receptor was correlated with cancerous angiogenic
property and cell mobility
– Unsaturated fatty acids, folic or, hyaluronic acids, Antibodies, etc.

88. Gene Delivery Slide 88 of 100
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Targets of siRNA in Cancer Therapy
 Cell Cycle
 Cell cycle progression is strictly controlled by numerous effectors
and checkpoints.
 Main effectors of cell cycle progression are cyclins and cyclin-
dependant kinases (cdk)
 Cyclins form the regulatory subunits and CDKs the catalytic
subunits of an activated heterodimer.
 When activated by a bound cyclin, CDKs performs phosphorylation
that activates or inactivates target proteins to orchestrate
coordinated entry into the next phase of the cell cycle.

89. Gene Delivery Slide 89 of 100
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Cell Cycle Control

90. Gene Delivery Slide 90 of 100
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Targets of siRNA in Cancer Therapy
 cyclin B1/cdk1 complex is the main effector implicated in G2/M transition.
Numerous studies have proven the overexpression of cyclin B1 in various
cancers as: oesophageal squamous cancer, non-small lung cancer, renal cancer,
prostate adenocarcinoma and breast cancer.
 Cyclin B1 siRNA inhibition strategy was employed in prostate and lung cancer in
vivo with peptide-based delivery systems
 Polo-like-kinase1(Plk1) plays key roles during mitosis notably in the
activation of cyclin B1/cdk1 complex
 Plk1 is overexpressed in multiple cancer types, including
melanoma, nonsmall cell lung cancer, colorectal cancer, and breast
cancer
 Plk1 siRNA delivery systems are being studied.

91. Gene Delivery Slide 91 of 100
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Targets of siRNA in Cancer Therapy
 Proliferation pathways
 Cancer cells are characterized by sustained proliferation resulting
from increased proliferative signals, decrease of negative feed-
back signals and replicative immortality acquisition.
 Cancer cells may produce or force their environment to produce
excessive growth factors like insulin growth factor (IGF), or
epidermal growth factor (EGF)
 Such external stimuli activate intracellular cascades like the MAPK
pathways or the phosphoinositide-3-kinase (PI3K)/serinethreonin
kinase AKT one, that promotes proliferation, division and survival
and were clearly described in cancerogenesis mechanism.

92. Gene Delivery Slide 92 of 100
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Targets of siRNA in Cancer Therapy
 Cell death and survival
 Pathways involved in cell death and survival have important role in
cancer development.
 Cancer cells display acquired or innate cell death resistance mechanisms
(to apoptosis or other cell death pathways).
 Angiogenesis
 Without angiogenesis, cancer could not grow up to more than 2 mm of
diameter.
 Vascular endothelium growth factors(VEGFs and VEGFA in particular)
are the most described secreted molecules implicated in angiogenesis of
human cancers.

93. Apoptotic
Pathways

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Some of Main References

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Some of Main References

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100. Thank You
for Your Kind Attention