Nucleic acid and cell based therapies involve gene therapy and cell therapy. Gene therapy aims to introduce new genes into cells to treat genetic diseases by replacing defective genes. Early gene therapy trials focused on ex vivo and in vivo approaches. Viral vectors like retroviruses were primarily used but posed safety risks. Non-viral methods using naked DNA or vectors like liposomes were developed. Diseases targeted include cancer, genetic disorders, and AIDS. Antisense oligonucleotides can bind mRNA and inhibit gene expression. RNA interference uses short interfering RNAs to induce sequence-specific gene silencing. Ribozymes and aptamers also provide nucleic acid based therapeutic approaches.

1. Nucleic acid and cell
based therapies

2. Gene Therapy
1980s and early 1990s Proteins of therapeutic
use
nucleic acid based therapeutics center around
gene therapy and antisense Tehnology
Early 2007 only three nucleic acid based
therapeutics gained worldwide approval.
 antisense-based product (Vitravene)
 aptamer (Macugen)
 gene therapy product(Gendicine)

3.  In cell based, fully differentiated cells
or groups of cells used
 organ or tissue transplantation

4. Principle of gene therapy
Stable Introduction of a gene into a genetic complement of
cell in such a way that expression of gene achieves a
therapeutic goal.
It is the replacement of defective gene with a new healthy
gene
It is a curative approach to inborn error of metabolism.

5. Some diseases for which gene
based clinical trials are in process

6. Basic approach to gene therapy

7. Vector systems used to deliver
genes into mammalin cells

9. Practical approaches that may be pursued when
undertaking gene therapy
In vitro
• In vitro gene therapy
entails removal of
target cells from the
body followed by
their incubation with
nucleic acid-
containing vector.
After the vector
delivers the nucleic
acid into the human
cells, they are placed
back in the body.
In vivo
• In vivo gene therapy
involves intravenous
administration of
the vector. The
vector has been
designed such that it
will only recognize
and bind the
intended target
cells. In this way, the
nucleic acid is
delivered exclusively
to those cells
In situ
• In situ gene therapy
entails direct
injection of the
vector immediately
adjacent to the body
target cells.

11. Some additional questions
choice of vector,
target cell and
protocol used
depends upon a
number of
consideration.
gene
therapy
treatment
protocol
used
choice of
target cells

12. Vectors used in gene
therapy

13. Types of vectors
Vectors capable of introducing genes
into recipient cells.
Viral based
Non –viral based

14. Retroviral vectors
Enveloped viruses.
Genome consists of ssRNA.
RNA transcribed and yields dDNA.
Integrates into host cell.

15. Structural genes in retroviral genome
• Code for viral protein.gag
• Codes for reverse transcriptase.pol
• Codes for viral envelope proteins.env

16. Other components
LTR harbours promoter
and enhancers.
promote integration
ψ packaging sequence.
viral RNA packaging

17. Retroviral genome
LTR Ψ gene of interest LTR

18. Retrovirus life cycle

19. Packing cells
Genetic material introduced
into cell
Recombinant contain
gag,pol,env and replication
deficient
One time,single hit gene
transfer system e.g
MoMuLv

21. Properties of Retrovirus
Integrate their proviral DNA into replicating
cells.
Efficiency of gene transfer in sensitive cells.
Long term,high level expression.
Integrates randomly.
Retro virus are promiscuous.
Complete copies are passed to daughter
cells.

22. Continue..
Good,high level stocks of
replication incompetent retroviral
particles.
Studies carried out in animal
species.

23. Advantages Disadvantages
Integrate into genome in a stable fashion Often damage during purification and
concentration.
Effectively enter into various cell types. Ability to infect only dividing cells.
Transduction efficacy. Lack of selectivity.
Fairly high level expression. Not infect all dividing cells.
Easy to propagate. Integrate randomly into the chromosome
of the recipient cells.

24. Drawback of retroviral gene
therapy
In 2002, Patients with SCID-X1 received with retroviral therapy
developed leukemic like condition
Due to the proviral integration at the site near the LM02 proto-oncogene promoter
leading to gene activation.
This resulted in initial ban on retro viral based gene therapy trials in some
world regions.

25. Adenoviral and
additional viral
vectors

26. Some other vectors are used for the purpose of gene
therapy in which includes
Adenoviruses
Adenoassociated viruses
Herpex virus
Sindbis virus

27. Adenoviruses
Relatively large
Non enveloped structure
Double stranded DNA
Large genome and complex

28. Adenoviruses have some
advantages or disadvantages
Advantages Disadvantges
Capable of gene transfer
to non dividing cells
High immunogenic in man
Easy to propagate Duration of tranferred
gene can vary
High gene expression Highly selective for cells

29. Adeno-associated virus
Small
Single stranded DNA
Need coinfecting
adenoviruses replicate

30. Small genes can be introduced into
adenoviral vector.
Facilitate long term gene expression

31. Herpes Simplex Virus
Herpes simplex virus
Neurotropic vector
Can deliver genes to PNS and CNS.
Upon infection Remains latent
in non dividing
neurons
Genome is in unintegrated form

32. Difficult to generate
viable herpes simplex
particle

33. Sindbis Virus
Member of
alphavirus
family
ssRNA
Simple
Infecting non
dividing cells

34. Formation of engineered sindbis virus

35. Manufacture of viral vectors

36. Non-viral vectors

37. •Use naked plasmid dna or dna
complexes as non-viral vector.
•Advantages:
• their low/non-immunogenicity;
•non-occurrence of integration of the
therapeutic gene into the host
chromosome

38. Methods of non-viral gene
delivery:
physical (carrier-free)
chemical approaches
(synthetic vector-
based gene delivery).

39. Naked DNA:
simplest method of non-viral transfection.
Clinical trials carried out of intramuscular injection of a naked DNA
plasmid have occurred .
low expression.

40. Lipoplexes:
 Plasmid DNA can be covered with lipids in
an organized structure like a liposome
complexed with DNA it is called a lipoplex
 3 types of lipids: anionic (negatively
charged)
 neutral
 cationic (positively charged)

41. Polyplexes:
Complexes of polymers with DNA are called
polyplexes
consist of cationic polymers and their
production is regulated by ionic interactions
polyplexes rapidly remove from circulation.
PEG attachment

42. cellular entry of (non-viral)
gene delivery:
Target: appropriate cell
surface
Therapeutic plasmid must
enter the cell and reach the
nucleus intact. Cellular
entry is generally achieved
via endocytosis.

43. Routes by which plasmid can reach
nucleus:
direct nuclear entry transport through
nuclear pores

44. manufacture of plasmid
dna:
Source
microorganism
fermentati
on
Cellular
recovery and
lysis
Removal of cell
debris
Plasmid
precipitation
Chromatographi
c purification
Concentratio
n,if required
Formulation
and
packaging

46. Genetic diseases?
A genetic disease is any disease that is caused
by an abnormality in an individual's genome.
Over 4000 diseases are characterized
Causes

47. Factors
Drug
compani
es
contribu
tion.
Expressi
on level
none of
the first-
generatio
n gene-
delivering
vectors
have
proven
fully
satisfactor
y.
Actual
genes
responsi
ble of
these
diseases
are
identifie
d..HGP.
Som
e
sho
ws
com
plex
ity

49. Example EX
VIVO GT
1990 – 4 year old Ashanti
DaSilva had a genetic
disorder called severe
combined
immunodeficiency (SCID)
Defect in ADA gene results in
an accumulation of dATP,
which is toxic to certain
types of T cells
Takes down the entire
immune system.

51. Example: IN-
VIVO GT
mutation on 7th chromosome.
Defective cystic fibrosis
transmembrane conductance
regulator (CFTR) gene.
Normally it serves as a pump
at the cell membrane to move
electrically charged chloride
atoms out of the cells
If cells can’t move chloride out,
they absorb water trying to
dilute the chloride in the cell
This leads to the production of
THICK sticky mucus

52. Vectors used to deliver CF gene to
airway epithelial cells:
1993 vector used: Adenovirus
1995 liposome
have potential to avoid critical problems immune
response, limited packaging capacity, and random
integration .
Liposomes may be mildly effective, but their
activity does not last.

54. Gene
therapy
and
cancer

55. 1.4 million cases reported.
50% survival rate using surgery,
chemo/radiotherapy

57. Low success rate due to:
1. requirement for improved, more target-specific vector
systems.
2. A requirement for a better understanding of how cancer cells
evade the normal immune response.
3. suffering from advanced and widespread terminal cancer
(i.e. little/no hope of survival if treated using conventional
therapies). Cancers at earlier stages of development will
probably prove to be more responsive to gene therapy.

58. Boosting the immune
response:
aim to boost the body's natural ability to
attack cancer cells.
Our immune system has cells that
recognize and kill harmful things that can
cause disease, such as cancer cells.
Involved introduction of TNF…

59. Steps:

60. Anti-cancer strategy:

61. Pro-drug
gene
therapy:

62. GENE THERAPY
AND AIDS

63. INTRODUCTION
Useful in treating medical condition
inherited disease,cancer and infectious
disease.
AIDS is a viral disease caused by intracellular
pathogens.
Different approaches used to treat AIDS

64. •Introduction of a
gene into pathogen
susceptible cells is
termed as
intracellular
immunization such
as introduction of a
gene into viral
sensitive cell coding
for an altered HIV
protein such as gag
tat env
•Mutant form of gag
is capable of
inhibiting viral
replication

65. The transfer to sensitive cell of a gene coding for
antibody fragments capable of binding to the HIV
enveolpe proteins this interfere with viral assembly
Recombinant cell have also been generated which
are capable of secreting soluble form of HIV cell
surface receptor like CD4 antigen
These soluble viral receptor would bind with
viriones

66. GENE BASED VACCINE
oAdministration of a DNA vector housing the gene
coding for a surface antigen protein from the target
pathogen
oAny body cell could be targeted.The target cell export
the resultant antigenic protein
oGene expression need only be transient to facilitate the
induction of an immune response
Gene based vaccine have entered for clinical trials
include malaria, hepatitis B and AIDS

67. Gene based vaccine have entered for clinical trials
include malaria, hepatitis B and AIDS

68. Causes Of Disease Occurrence
the expression of oncogenes
overexpression of cytokines
overproduction of angiotensinogen Hypertension

69. Antisense Technology
Definition
The specific sequence of nucleotide bind to mRNA or
DNA when the sequence is known that will cause a
specific disease. This leads to gene turning off

70. Antisense technology
Antisense Nucleotides
Short and single stranded stretches of Nucleic acid having a
specific sequences of nucleotides.
Oligonucleotide binds to DNA or mRNA.
Binding prevents/block the Transcription and Translaion process

72. Antisense oligonucleotides and
their mode of action
Identify the suitable sequences of mRNA
Blind or shot gun used to
designed the
oligonucleotides
Computer programs For
mRNA sequencing and its
accessible region for
oligonucleotides

73. Antisense oligonucleotides
and their mode of action

75. Advantages of Oligos
It acts as therapeutic agent in
Cancer
Viral diseases such as HIV,hepatitis B
Herpes and Papilloma Infection

76. Example Cancer
BCL2 gene
These gene increase the
neoplastic progression
Inhibit Cell apoptosis
Survivin gene
Present in cancer
patient.
Cell division and
apotosis inhibiton.
Lung ,colon,breast and
prostate cancer.
Clusterin genes
Codes for cytoprotective
chaperon protein.
Upregulation results in
various human cancer.

77. Advantages
Low
Toxicity
Required in
minute
quantity as
mRNA is
present in
nanomolar
concentratio
n
Easily
Formed by
automatic
synthesizer

78. Disadvantages
sensitivity
to
nucleases
very low
serum
half lives
poor rate
of cellular
uptake
orally
inactive.

79. Oligonucleotide pharmacokinetics
and delivery
Intravenous administration
Sub cutaneous administration
Intra dermal administration
Charged Oligo's enter in cell by receptor mediated
endocytosis
UnCharge Oligos enter in the cell through Passive
diffusion and endocytosis

80. Continue
Liposomes mediated and polymeric carriers are used
for the delivery of oligos
Oligos appear to be ultimately metabolized within the
cell by the action of nucleases, particularly 3´-
exonucleases. Breakdown metabolic products are then
mainly excreted via the urinary route.

81. Phosphodiester linkage

82. Manufactureofoligos

83. Oligonucleotides
Oligonucleotides are
short DNA or RNA molecules, oligomers, that
have a wide range of applications in genetic
testing, research, and forensics.
Commonly made in the laboratory by solid-
phase chemical synthesis.

84. Oligonucleotides
“Oligo” – Prefix meaning few (~ 2-10).
Nucleosides can be obtained from either
natural sources (i.e. salmon sperm) or
chemically synthesized.
We use phosphoramidite method for
manufacturing oligos.

85. PhosphoramiditeMethod
Developed in 1980.
The principle of this method was developed
by McBride and Caruthers in 1983.
Currently considered as the standard
synthesis.
Used in most automated synthesizers today.

86. Cycle
The cycle consists of four steps:
1. De-protection
2. Coupling
3. Oxidation
4. Capping

87. De-protection
Trityl group attached to the 5’ carbon of the
pentose sugar of the recipient nucleotide is
removed by trichloroacetic acid (TCA) leaving
a reactive hydroxyl group.

88. Coupling
The phosphoramidite monomer is added in the
presence of an activator such as a tetrazole
This structure then reacts with the hydroxyl group
of the recipient and the 5’ to 3’ linkage is formed.
Tetrazole = Class of synthetic organic heterocyclic
compound, consisting of a 5-member ring of four
nitrogen atoms and one carbon atom. The simplest
is tetrazole itself, CH₂N₄. They are unknown in
nature.

89. Oxidation
The oxidation step stabilizes the phosphate
linkage in the growing oligonucleotide.
The traditional method of achieving this is by
treatment with iodine in water.

90. Capping
Any remaining free 5’-hydroxyl groups are
blocked at the capping step in an irreversible
process.

92. After having synthesized the full length sequence,
the oligonucleotide is then released from the solid
support using a base, such as aqueous ammonia or
a mixture of ammonia and methylamine. This will
also remove protection groups from the
nucleobases.
The oligonucleotide is now ready for purification.
oligonucleotide is purified with RP-HPLC where the
retention time is to a large.

93. RNA interference and
ribozymes
RNAi represents a sequence-specific post-
translational inhibition mechanism of gene
expression,
Induced ultimately by dsRNA
Entry of dsRNA triggers its cleavage into short
(21–23 nucleotide long) sequences called short
interfering RNAs

94. This cleavage is catalyzed by a Enzyme
known as Dicer
RNA inducedsilencing complex (RISC)
antisense’ siRNA strand then facilitates RISC
binding to a specifi c mRNA via Watson–Crick
base complementarity, which is then
degraded by RISC nuclease activity.

96. Ribozyme
RNA sequences can function as
catalysts. This is known as Ribozymes
Many ribozymes will cleave
Target mRNA where there exists a
particular triplet nucleotide sequence
G–U–C

97. Ribozyme

98. Aptamers
Aptamers are single-stranded DNA or RNA-based
sequences allowing them to bind a specific
target molecule.
Identification of specific aptamers binding the
target molecule
SELEX (systematic evolution of ligands by
exponential enrichment)

99. affinity-based purifi cation,
target validation
drug discovery, diagnostics and
therapeutics.

100. low immunogenicity
In order to prevent renal removal, aptamers
are usually conjugated to PEG.
Their half-lives can most effectively be
extended via chemical modification