The document discusses nucleic acid based therapeutic delivery systems for gene therapy. It begins with an introduction to nucleic acid therapeutics including DNA and RNA. It then discusses various mechanisms for inhibition of gene expression, such as antisense mechanisms, RNA interference, aptamer selection, and ribozymes/DNAzymes. It also discusses various physical methods for targeting nucleic acids, including gene guns, naked DNA injection, hydrodynamic delivery, electroporation, photodynamic therapy, magnetoporation, and ultrasound. The document concludes with sections on gene therapy, types of gene therapy including germline and somatic gene therapy, and methods of gene therapy delivery.

1. Nucleic Acid Based Therapeutic Delivery System :
Gene Therapy
Submitted to
Dr. Vivek Dave
Associate Professor
Head of Department
Department of Pharmacy
School of Health Science
Presented by
Mr. Sree Prakash Pandey
(CUSB2006122010)
M.Pharm (Pharmaceutics)
1st year (II Sem)
School of Health Science
1
Central University of South Bihar, Gaya

2. 2
• Nucleic Acid Therapeutics
• Nucleic acid, DNA & RNA
• What is Gene expression & regulation ?
• Different mechanism for inhibition of Gene expression
• Physical targeting of nucleic acids
• Gene therapy
• Types of Gene Therapy
• Methods of Gene Therapy
• Polymers and lipids in gene delivery
• Application
• Conclusion
• References
CONTENTS

3. 3
Nucleic Acid Therapeutics

4. • The process of gene delivery and expression is known as
transduction.
• Therapeutic NAs comprise DNA (oligonucleotides, plasmid,
viruses, artificial chromosomes, and bacteria) and RNA
(oligonucleotides, ribozymes, siRNA, mRNA, and viruses).
• The exogenous nucleic acids can be used to modify gene
expression.
• Oligonucleotides as therapeutics for manipulating gene
expression in living cells.
4
Nucleic Acid Therapeutics
Oligonucleotides
is a
polynucleotide
whose
molecules
contain a
relatively small
number of
nucleotides.

5. Cont…
• Synthetic oligonucleotides (ODNs) are short (<30 nucleotides) nucleic acid
strands (DNA or RNA), which are chemically synthesized.
• Vectors that have been developed to overcome these obstacles fall into two
broad categories: nonviral and viral vectors.
• The nonviral vectors consist of naked DNA delivered by injection, liposomes
(cationic lipids mixed with nucleic acids), nanoparticles, and other means.
• Viral vectors are derived from viruses with either RNA or DNA genomes and
are represented as both integrating and nonintegrating vectors.
• Non-viral methods are preferred due to their low pathogenicity.
5

6. Nucleic acid, DNA & RNA
• Nucleotides consist of a nitrogenous base, a
pentose and a phosphate.
• Nucleic acids are the polymers of nucleotides
(polynucleotides) held by 3' and 5' phosphate
bridges.
• There are two types of nucleic acids, namely
deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA).
6

7. Cont…
• The pentose sugar is D-ribose in ribonucleotides of RNA while in
deoxyribonucleotides (deoxynucleotides) of DNA, the sugar is 2-deoxy
D-ribose.
• Primarily, nucleic acids serve as repositories and transmitters of genetic
information.
• DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides).
• lt is composed of monomeric units namely deoxyadenylate (dAMP),
deoxyguanylate (dGMP), deoxy-cytidylate (dCMP) and
deoxythymidylate (dTMP).
7
Reference: Satyanarayana U. et al 2006

8. • RNA is a polymer of ribonucleotides
held together by 3',5'-phosphodiester
bridges.
• The three major types of RNAs with
their respective cellular composition
are given below
1. Messenger RNA (mRNA) : 5-10%
2. Transfer RNA (tRNA) : 10-20%
3. Ribosomal RNA (rRNA) : 50-80%
8

9. 9
What is Gene expression & regulation ?
 Gene expression is the process by which the information
encoded in a gene is used to direct biosynthesis of a protein
molecules that produces the structure of cell.
 The process of gene expression involve two main stages-
Transcription
a process in which
ribonucleic acid (RNA) is
synthesized from DNA.
Translation
the biosynthesis of a protein
or a polypeptide in a living
cell.
Gene is the
basic
physical and
function unit
of heredity
which is
made up of
DNA & store
genetic
information.

10. Cont…
• Gene regulation is the process of turning genes on and off.
• It is a label for the cellular processes that control the rate and manner of gene
expression.
• A complex set of interactions between genes, RNA molecules, proteins
(including transcription factors) and other components of the expression
system determine when and where specific genes are activated and the
amount of protein or RNA product produced.
10
Reference: Satyanarayana U. et al 2006

11. 11
Ribozymes and DNAzymes
Antisense mechanism
Aptamer selection
RNA interference
Antigene mechanism
The inhibition of gene expression is based on following mechanism-
Different mechanism for inhibition of Gene expression

12. 12
Antigene
therapy involves
blocking of
transcription (by
antigene
oligonucleotide).
1. Antigene mechanism

13. Cont…
• In this mechanism, the Triplex-helix forming oligonucleotides are synthetic
single-stranded DNA, which hybridize to purine or pyrimidine-rich region in
the major groove of double-helical DNA through Hoogsteen base pairing. If
a stable triple helix is formed, it prevents unwinding of double helical DNA
necessary for transcription of the targeted region or blocks the binding of
transcription factor complexes.
• This mechanism is not considered efficient for clinical applications even
though it provides an opportunity for therapeutic interventions at a very early
stage.
13

14. 14
The antisense
therapy,
translation is
inhibited (e.g
antisense
oligonucleotide).
2. Antisense mechanism

15. Cont…
• In the antisense mechanism, the Reverse-complementary
oligonucleotides, which hybridize to the mRNA strand of the
targeted gene.
• After hybridization, antisense oligonucleotides block expression
either sterically by obstructing the ribosomes or by forming an
RNA-DNA hybrid, which is a substrate for RNase H enzyme,
thereby causing the cleavage of target mRNA.
15

16. 16
RNA interference (RNAi)
or post-transcriptional gene
silencing is a natural
process in eukaryotic cells
by which double-stranded
RNA targets mRNA for
cleavage in sequence-
specific manner.
3. RNA interference

17. Cont..
 The mechanism of RNAi involves processing of a very long (500– 1000
nucleotides) double-stranded RNA, which is cleaved into short double-
stranded RNA by the DICER enzyme.
 DICER, also known as endoribonuclease Dicer or helicase with RNase
motif, is an enzyme that in humans is encoded by the DICER1 gene.
 Dicer facilitates the activation of the RNA-induced silencing
complex (RISC), which is essential for RNA interference.
17

18. 18
Aptamers are nucleic
acid ligands (15–40
nucleotides) isolated
from combinatorial
oligonucleotide
libraries by in vitro
selection.
4. Aptamer selection

19. Cont…
• In solution, the oligonucleotide chain forms intramolecular interactions
that fold the molecule into a complex 3D shape.
• Aptamers have capability to tightly and specifically bind to the target
molecules ranging from small molecules to complex multimeric
structures.
• The therapeutic potential of aptamers arises from the fact that many
aptamers targeted against proteins are able to interfere with their
biological activity.
19

20. • Other nucleic acid agents such as microRNA
are also known and so are nucleic acids with
catalytic activity: ribozymes and DNAzymes.
• Ribozymes are RNA molecules containing
different catalytic motifs. They hybridize (bind)
to the substrate RNA through Watson–Crick
base pairing and cause sequence-specific
cleavage.
• DNAzymes are DNA molecules containing a
catalytic motif and similar to ribozyme, cleave
the substrate RNA after binding to it.
20
5. microRNA : Ribozymes and DNAzymes

21. Different mechanisms for selective inhibition of gene expression
21
RISC:- RNA-
induced silencing
complex
siRNA :- small
interfering RNA

22. 22
Complexatio
n
• The assembly of complexes between the gene carrier
and the nucleic acid molecules
Condensatio
n
• pDNA is condensed into sub-micron particles with an
overall positive surface charge
Binding
• This allows binding to the negatively charged cell
surface and promote subsequent uptake via endocytosis.
Overview of the transfection pathway

23. 23
Destabilizati
on
• Release into the cytosol is facilitated by destabilization of endosome
membrane with pH-responsive components of the gene carrier.
Transcripti
on
• The pDNA must be imported into the nucleus for transcription.
Decondensa
tion
• Transcriptional activity of the transgene is favored by intranuclear
disposition within the euchromatin domain, as well as efficient de-
condensation from the gene carrier.
Replication
&
Retention
• Long-term expression of the transgene will require replication and nuclear
retention of the pDNA as well as avoiding the transgene clearance activity
of the immune response.
23

24. 24
Schematic overview of the transfection pathway

25. Physical forces such as electric or magnetic field, light, hydrodynamic pressure
or mechanical forces are used in some strategies for NAs delivery, such as
25
Targeting
of NAs
Naked
DNA
Injection
Hydrodyn
amic
Delivery of
NAs
Electropor
ation
Jet
Injection
Ultrasound
(US)
Gene Gun
Photochem
ical
Internaliza
tion (PCI)
PHYSICAL TARGETING OF NUCLEIC ACIDS

26. • A gene gun is a device used to delivery of
exogeneous DNA, RNA or protein to target
cell. This method is called Biolistics.
• Gene gun or Particle-mediated transfection
(PMT) is a useful approach of non-viral gene
transfer in cancer therapy.
Application
• DNA vaccination and cytokine gene
therapy. 26
1. GENE GUN

27. 27
Advantages
 Gene gun administration of DNA is
safer than parenteral injection.
 Gene gun-based DNA vaccination is
much more efficient than parenteral
injection, and therefore the total
amount of DNA required for
delivery is less.

28. • The main advantage of this method is
its simplicity, involving direct plasmid
injection, but results in low levels of
gene transfer.
• Several reports on direct injection to
various tissue sites have been
documented with one example of this
method being the intra-tumoral
delivery of a cytosine-deaminase
suicide gene. 28
2. Naked DNA Injection

29. • Hydrodynamic delivery employs the force
generated by a relatively rapid injection of a
large volume of solution into the
bloodstream.
• This approach introduces naked plasmid
DNA into cells in highly perfused internal
organs such as liver with a remarkable
efficiency.
29
3. Hydrodynamic delivery
• Gene delivery efficiency in this method is a function of factors which include
the anatomic structure of the organ, the injection volume, and the speed of
injection.

30. Cont…
• A large variety of substances with different molecular weights and
chemical structures such as small dye molecules, proteins,
oligonucleotides, small interfering RNA, and linear or circular DNA
fragments applied to intracellular delivery of any water-soluble
compounds, small colloidal particles, or viral particles directly into
cytoplasm without Endocytosis.
• This method is successful for liver transfection.
30

31. • The Jet injector is a device narrowed with high pressure of
liquid stream that penetrates the epidermis of skin to target
tumor/cancerous cell.
• In the jet injection method, mechanical compression is used to
move fluid, including DNA, through a small orifice rapidly, in
less than a second. The result is a high pressure stream
penetrating tissue without using any needle for particles.
• Recent studies show that this approach can efficiently express
genes in the tumors and be applied for cancer therapy using
minimum naked DNA. 31
4. Jet injection method

32. 32
USED
 for gene delivery into large muscles
 for gene transfer into tumors,
 for cardiac gene therapy.

33. • In this method, short-duration electric field
pulses are applied to various tissues such as
muscle, skin, liver, lung, and tumor after
local administration of NAs to overcome
the barrier of the cell Membrane.
• Injection of DNA followed by local
electroporation in tumors enhances gene
transfer into cells applicable in delivering
of a large variety of molecules and
materials such as ions, drugs, dyes, tracers,
antibodies, oligonucleotides, RNA and
DNA, both in vivo and in vitro.
33
5. Electroporation

34. • Photodynamic therapy is an emerging
technique which can be applied efficiently for
NA delivery to a specific light-exposed site.
• Photochemical internalization (PCI), in
which photosensitizing compounds localized
in endocytic vesicles can be excited by the
light in a specific wavelength and initiate
photochemical reactions.
34
6. Photodynamic therapy
OPTOPORATION

35. 35
6. Magnetoporation
Magnetoporation
is to deliver
the nucleic
acids into the
cell under the
influence of
magnetic field
with
transfecting
plasmid DNA int
o a variety of
tumor cells.

36. • This approach facilitates the intra-tumoral injected
DNA delivery into tumor tissues and gene transfer
at cellular and tissue levels
• The duration of US treatment, and the amount of
plasmid DNA used are important to transfection
efficiency of this method
• The vehicles such as gas-filled poly(D,L-lactide-
coglycolide) (PLGA) microparticles were
systemically used to encapsulate plasmid DNA
and US was applied to destruct the gas core and
release the plasmid at the target site 36
7. Ultrasound (US)

37. 37
GENE THERAPY

38. GENE THERAPY
HISTORY OF GENE THERAPY
• The term gene therapy was first introduced at the International Congress of
Genetics.
• The techniques utilized by Gregor Mendel in the 1850s, which were then developed
by Ronald Fischer at the beginning of the 20th century, formed the turning points in
genetics. The work of both Mendel and Fischer laid the foundations of genealogy.
• The material they studied was later termed as gene by the Danish botanist Wilhelm
Johannsen.
• The first gene therapy trial in humans was conducted at the beginning of the 1970s,
and it was observed that naturally occurring DNA and RNA tumor viruses
successfully delivered new genetic information to the genomes of mammal cells.38

39. • Gene therapy is a novel form of molecular medicine
that has the potential to influence significantly human
health.
• It promises to provide new treatments for the large
number of inherited and acquired diseases.
• Gene therapy is based on the substitution or
replacement of a defective gene with a functional
copy or manipulation of a gene function or expression
using short nucleic acids (NAs).
39
Gene
therapy
is the
process of
inserting
genes
into cells
to treat
diseases.
DEFINITION
GENE THERAPY

40. Cont…
Gene-based therapy is commonly associated with gene augmentation or gene
replacement therapy, where the deficient gene product is supplied with a
functional version.
Gene augmentation
• Gene augmentation consists on the delivery of a new protein-coding gene to treat
a disease.
• In gene augmentation therapy (GAT), the aim of the treatment is to enhance the amount of
protein product by delivering exogenous nucleic acid molecules
• The exogenous nucleic acid molecules containing instruction for the expression of the
deficient protein.
• GAT has been applied to cancer therapy via de novo expression of suicide genes that have
the ability to induce cell death on its own expression and can range from apoptosis
inducer, such as caspases to enzymes that converts prodrugs to cytotoxic compound
40

41. 41
Gene knockdown
• An experimental technique by which the expression of one or more of
an organism's genes is reduced.
• The reduction can occur either through genetic modification or by treatment
with a reagent such as a short DNA or RNA oligonucleotide that has a
sequence complementary to either gene or an mRNA transcript.
Gene repair
• Targeted gene repair is a technique used to correct a mutation at a specific site
in an episome or chromosome.
• It uses synthetic oligonucleotides together with the cell's inherent DNA
repair system to direct single base pair changes.
Cont…

42. 42
Gene Therapy
Germline
Gene Therapy
Somatic Gene
Therapy
Types of Gene Therapy

43. Germline Gene Therapy
• In germline gene therapy (GGT), germ
cells such as sperm or egg cells are modified by
the introduction of functional genes into their
genomeas.
• Modifying a germ cell causes all the organism's
cells to contain the modified gene.
• The change is therefore heritable and passed on
to later generations.
43
There is the
introduction of DNA
into germ cells is
passed on to the
successive generation
which treat the
disease.

44. Somatic Gene Therapy
• The genetic alterations in somatic cells
are not carried to the next generation.
• Therefore, somatic cell gene therapy is
preferred and extensively studied with
an ultimate objective of correcting
human disease.
44
There is the insertion
of a fully functional
and expressible gene
into a target somatic
cell to correct a
genetic disease
permanently.

45. 45
Can be applied to
only selected
tissue, e.g, bone
marrow cells
Cells modification
are carried outside
from the patient
body and then
transplanted back
Ex-vivo
Gene
therapy
The direct delivery
of therapeutic gene
into the target cells
of patient
Cells modification
are carried inside
to the patient body
and then
transplanted back
In-vivo
Gene
therapy
Method of Gene Therapy

46. Steps of Ex-vivo Gene therapy
• The following steps involve to describe the technique of ex-vivo gene therapy-
46
Transplant the modified cells to the patient.
Select the genetically corrected cells (stable
transformants) and grow.
i.Introduce the therapeutic gene to correct
gene defect.
i.Grow the cells in culture.
i.lsolates cell with genetic defect from a
patient.

47. Cont…
VECTORS IN GENE THERAPY
• The carrier particles or molecules used to deliver genes to somatic cells are
referred to as vectors.
• Viruses - particularly Retroviruses, RNA is the genetic material in
retroviruses. As the retrovirus enters the host cell, it synthesizes DNA from
RNA (by reverse transcription).
• Human Artificial Chromosome (HAC) - is a synthetic chromosome that can
replicate with other chromosomes, besides encoding a human protein.
• Bone marrow cells - contains totipotent embryonic stem (ES) cells. These
cells are capable of dividing and differentiating into various cell types.
• Ex-vivo gene therapy e.g. Sickle-cell anemia, SCID, Thalassemia, etc.
47

48. In-vivo Gene therapy
• The direct delivery of the therapeutic gene (DNA) into the target cells of a particular
tissue of a patient.
• Several tissues like liver, muscles, skin, spleen, lung, brain and blood cells.
• Gene delivery can be carried out by viral or non-viral vector systems.
48
Viral systems
Many viral vector systems
have been developed for
gene delivery.
These include retroviruses,
adenoviruses, adeno-
associated viruses and herpes
simplex virus.
Non-viral
vector systems
The viral proteins often
induce inflammatory
responses in the host. So,
non-viral vectors proposed.
Pure DNA, Lipoplexes,
Human artificial
chromosomes, etc.

49. Process of In-vivo Gene therapy
49

50. 50
POLYMERS AND LIPIDS IN GENE DELIVERY
Polyplexes
 Polyplexes are formed when cationic polymers interact with
DNA through electrostatic interactions.
 Complexation or encapsulation of nucleic acids by the positively
charged polymer reduces the charge of the nucleic acid and
forms particles in the nano-size range, resulting in increased
permeability.
 Chitosan, poly-L-lysine and polyethylene amine (PEI) are
cationic polymers widely used for gene delivery.
 Polyplexes having low transfection ability or lacking specificity
for a target could be made more efficient by modifying the
carrier.

51. 51
Lipoplexes
 In a similar manner of polyplex, the lipids with a positive
charge are able to form complexes with DNA.
 Such liposomes or lipoplexes have better transfection
ability than neutral liposomes.
 Positively charged liposomes are able to neutralize the
negative charge on DNA and nucleic acids. Due to the
lipidic nature of the carrier, the transfection ability of
lipoplexes are generally high.
New cationic liposomes as vectors which form lipoplexes with plasmid DNA
have been developed for gene delivery and have shown high transfection
efficiency after systemic administration.

52. 52
SCHEMATIC REPRESENTATION FOR DELIVERY OF LIPOPLEXES

53. 53
Nanoplexes
 Nanoparticles formed by dispersion of nucleic acids in polymers
as nanospheres and encapsulation of DNA by polymers such as
chitosan, albumin or collagen to form nano-capsules are
categorized as nanoplexes.
 The entrapment of genetic material into nanoparticles constitutes a
promising approach for increasing the in vitro and in vivo
transfection activity.
 Calcium phosphate-DNA co-precipitates have been used for gene
delivery for more than 35 years. They are nontoxic, bioresorbable,
easy to produce, and able to complex with the negatively charged
phosphate ions of nucleic acids by interaction through calcium
ions.
 The use of poly(ethylene glycol) functionalized bisphosphonate (PEG-bp)
produced stabilized calcium phosphonate-DNA nanoparticles which had low
toxicity and sustained ability to transfect cells.

54. 54
1. Applications of Gene Therapy in Cancer
• The three conventional modalities of treatment of cancer surgery,
radiotherapy, and chemotherapy are often unsuccessful in treating cancer.
• Therefore, Gene therapy is the emerging fourth modality for treatment of
cancer.
• Various types of cancer are now active targets for gene therapy clinical trials.
Prostate Cancer
Breast Cancer
Lung Cancer
Brain Cancer
Application of Gene therapy

55. 55
2. Applications of Gene Therapy in Cardiovascular(CV) Diseases
• Gene therapy is specifically important for monogenetic CV disorders like
hypertension or hypercholesterolemia.
• The primary goal of gene therapy is to insert the corrected form of the
mutated gene into the appropriate target cell type to restore normal function.
Hypertension
Hypercholesterolemia and Atherosclerosis
Thrombosis
Ischemia

56. • Gene therapy is specifically important for monogenetic CV disorders like
hypertension or hypercholesterolemia.
• The primary goal of gene therapy is to insert the corrected form of the
mutated gene into the appropriate target cell type to restore normal function.
 Hypertension
 Hypercholesterolemia and Atherosclerosis
 Thrombosis
 Ischemia
56

57. 57
3. Applications of Gene Therapy in Kidney Diseases
Glomerulonephritis and Renal Fibrosis
• Glomerulonephritis is characterized by mesangial cell proliferation,
extracellular matrix accumulation, and renal fibrosis leading to renal failure.
• A strategy was designed to target mesangial cell growth using appropriated
AS-ODN against platelet-derived growth factor (PDGF) and transforming
growth factor- β (TGF-β) genes, which are responsible for mesangial cell
growth.
Renal Transplantation
• The immune response of a host’s body to renal graft is the major problem in
kidney transplantation.
• Gene therapy can provide an alternative therapy to reduce immune response
in renal transplantation.

58. 58
4. Applications of Gene Therapy in Cystic Fibrosis
• Cystic fibrosis (CF) is one of the most common chronic progressive and
frequently fatal autosomal recessive genetic diseases affecting the body’s
exocrine glands. CF is caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene, located on the long arm of chromosome 7.
The normal CFTR gene encodes for formation of chloride channel protein,
responsible for salt balance across the cell membrane.
• In CF patients, CFTR is unable to perform the normal functions, leading to
dehydration of affected organs such as the sweat glands, lungs, pancreas, and
digestive system. Because lungs are the primary organs affected in CF patients,
pulmonary symptoms are responsible for the majority of mortalities.
• Gene therapy of CF involves the insertion of a normal copy of the CFTR gene
into affected cells, which could potentially correct the biochemical abnormality
of these cells and thus prevent or ameliorate disease in that organ.

59. 59
5. Applications of Gene Therapy in Hemophilia
• A deficiency or dysfunction of either FVIII or FIX leads to bleeding
following vascular injury. Hemophilia is an X-linked inherited bleeding
disorder characterized by severe uncontrolled hemorrhagic episodes that can
even be fatal.
• Hemophilia A and B resulted from a subnormal level or deficiency of
essential cofactors, FVIII and FIX, respectively.
• The defect in FVIII and FIX synthesis occurs due to mutation in the genes
synthesizing them, respectively.
• Currently, hemophilia is treated with protein replacement therapy using either
plasma-derived or recombinant coagulation factors.
• In a clinical trial, the nonviral method was used for the delivery of plasmid
containing FVIII cDNA in patients with severe hemophilia, resulting in 1–2%
expression of FVIII in six patients .

60. 60
6. Applications of Gene Therapy in Gaucher Disease
• Gaucher disease (GD) is an inherited, autosomal recessive, most common
lysosomal storage disorder characterized by a decreased level of enzyme β-
glucosidase (glucocerebrosidase).
• Enzyme replacement therapy (ERT) is the standard treatment for GD.
However, it is very expensive to administer biweekly infusions of the
recombinant enzyme throughout the life of the patients.
• Gene therapy could provide an alternative for GD by delivering the
glucocerebrosidase (GC) gene using vectors ex vivo and in vivo. In a study,
insertion of the human GC cDNA into mouse fibroblasts resulted in the
appearance of human enzyme activity.

61. Conclusion
• There are a number of human diseases, both genetic and acquired, for
which gene based therapeutic approaches are gaining appraisals for
genetic and acquired diseases.
• Gene therapy has been proven to be an incredibly powerful tool for the
prevention and cure of diseases as it aims to treat the cause rather than
the symptoms of diseases.
• A gene medicine is composed of an expression system with a gene of
interest and a gene delivery system.
61

62. 62
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