NUCLEIC ACID BASED

THERAPEUTIC DELIVERY SYSTEM

1
U.SAHUL HAMEED NIYAZ
DEPT.OF PHARMACEUTICS
MMC,CHENNAI-03.
2 CONTENTS

 GENE EXPRESSION SYSTEM

 GENE TRANSFER TECHNOLOGIES
MECHANICAL AND ELECTRICAL TECHNIQUES
VECTOR –ASSISTED DELIVERY SYSTEMS
 VIRAL AND NON VIRAL GENE TRANSFER
 LIPOSOMAL GENE DELIVERY SYSTEMS
 REFERENCES
3 GENE EXPRESSION SYSTEM

INTRODUCTION:-
Gene expression is a process by which a genes information is
converted into the structures and functions of a cell by a process of
producing a biologically functional molecule of either protein or
RNA (Gene product) is made.
Gene expression is assumed to be controlled at various points in the
sequence leading to protein synthesis.
Protein synthesis is the process in which cells build protein from
information in DNA in two major steps:
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Transcription :
Synthesis of an RNA that is complementary to one of the strands of
DNA according to instruction stored along a specific sequence ( a
gene) of a DNA molecule.

Translation:
Ribosomes read a messenger RNA and make protein according to its
instruction.
5 GENE TRANSFER TECHNOLOGIES

 Gene transfer technologies can be classified into three general types

Electrical Techniques
Mechanical Transfection
Vector –Assisted Delivery Systems
6 MECHANICAL AND ELECTRICAL
TECHNIQUES
 Introducing naked DNA into cells by mechanical and electrical techniques
include microinjection, particle bombardement ,the use of pressure and
electroporation.
 Microinjection is highly efficient since one cell at a time is targeted for
DNA transfer, but it is time consuming.
 Ballistic transfer of gold microparticles maybe performed using particles
bombardment equipment such as the gene gun.
 Electroporation is achieved using high-voltage electrical current to
facilitate DNA transfer that results in high cell mortality and is not suitable
for clinical use.
7 VECTOR ASSISTED DELIVERY SYSTEMS

 Vector assisted DNA /gene delivery systems can be classified into two
types based on their origin:

Biological Viral DNA Delivery systems

Chemical Non-Viral delivery systems.
8 VIRAL DELIVERY SYSTEM

 In viral delivery systems, non-pathogenic attenuated viruses can be used as

delivery system for genes /DNA molecules, especially plasmids.
 These viral DNA delivery vectors include both RNA and DNA viruses.
 The viruses used for gene therapy vectors can be classified in four types
Retroviruses
Adenoviruses
Adeno Associated Viruses
Herpes Simplex Viruses
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 Gene expression using viral vectors has been achieved in tissues such as
kidney ,heart muscle, eye and ovary.
 Gene therapy using viral system has been made considerable progress for
the treatment of a wide range of diseases, such as muscular dystrophy,
AIDS and cancer viruses are used in more than 70% of human clinical gene
therapy trials world wide.
 The only approved gene therapy treatment (Gendicine) delivers the
transgene using a recombinant adenoviral vector.
 DNA delivery using viral vectors has been extensively reviewed.
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 The first-generation retroviral vectors were largely derived from

oncoretroviruses, such as the Moloney Murine Leukemia virus (MMuLv),
and were unable to transfer genes into nondividing cells.
 This limited the potential for their application as a delivery system in gene
therapy.
 The utilization of the lentivirus family of retroviruses has overcome this
shortcoming.
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 Lentiviruses, which include Human immunodeficiency virus type 1

(HIV-1), Bovine immunodeficiency virus (BIV), Feline
immunodeficiency virus (FIV), and Simian immunodeficiency virus
(SIV), are able to transfer genes to nondividing cells.
 Retroviral vectors used in gene therapy are replication deficient, such that
they are unable to replicate in the host cell and can infect only one cell.
 This characteristic, although essential for the safety of viral vectors in gene
therapy, imposes restrictions on the amounts of virus that can safely be
administered.
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 Retroviral-mediated delivery of therapeutic DNA has been widely used in

clinical gene therapy protocols, including the treatment of cancers, such as
melanoma and ovarian cancer, adenosine deaminase deficiency–severe
combined immune deficiency, and Gaucher’s disease.
 Retroviral vectors are capable of transfecting high populations (45–95%) of
primary human endothelial and smooth muscle cells, a class of cells that are
generally extremely difficult to transfer.
 Adenoviruses have been used to deliver therapeutic DNA to patients
suffering from metastatic breast, ovarian, and melanoma cancers.
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 Indeed, the severe immune response of the host contributes to the limited
survival of the adenoviral DNA in the targeted cells and results in a
transient expression of the therapeutic gene since the adenoviral DNA is
lost overtime.
 First-generation adenoviral vectors were able to accommodate the
introduction of therapeutic genes over 7 kb long (but rarely larger) into
targeted cells.
 However, the generation of gutless adenoviral vectors, which lack all viral
genes, has facilitated adenoviral delivery of up to 30 kb of a therapeutic
DNA sequence with decreased toxicity.
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 Adenoviral mediated gene transfer in COS-7 cells was significantly higher

than that achieved by liposomal delivery systems.
 The use of Adeno-Associated Viral (AAV) vector provides an alternative to
adenoviral vectors for gene therapy and a means for long-term gene
expression with a reduced risk of adverse reactions upon administration of
the vector.
 AAV viruses are linear, single-stranded DNA parvoviruses that are not
associated with any disease in humans.
 In humans, the site of AAV viral DNA integration is on chromosome 19.
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 In the engineering of AAV vectors, most of the AAV genome can be

replaced with the therapeutic gene, which significantly reduces potential
adverse responses of the host to viral infection.
 However, the size of the therapeutic gene is limited to approximately 5 kb.
 First-generation adeno-associated viruses had a very small capacity of ~4.7
kb for encapsulation of the plasmid DNA cargo.
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 Recent reports demonstrate efficient production of second-generation

Adeno-Associated Viruses with higher encapsulating capabilities.
 It has been demonstrated that adenoviruses in formulations may lose their
potency after storage in commonly used pharmaceutical vials.
 Herpes Simplex Virus (HSV) vector is a large and relatively complex
enveloped, doublestranded DNA virus that has the capacity to encode large
therapeutic genes and, like AAV, can remain latent in infected cells,
providing the potential for long-term expression of the therapeutic gene.
 Although able to infect many cell types, HSV vectors currently are limited
in their use by vector toxicity.
17 NON-VIRAL DELIVERY SYSTEM

 Non-viral delivery systems have the greatest advantage over viral delivery
systems-the lack of immune response and ease of formulation and assembly.
 Commonly used Non-Viral vectors for delivery of DNA-based therapeutics
can be classified into three major types:
Naked DNA delivery systems,
Polymeric delivery systems,
Liposomal delivery systems.
18 NAKED DNA DELIVERY SYSTEM

 Naked DNA can be administered via two possible routes, either by ex vivo
delivery or by in-vivo delivery.
 The ex vivo method of naked DNA delivery has been used successfully for
the introduction of DNA into endothelial and smooth muscle cells ; its
reliance on the culture of harvested cells renders it unsuitable for many cell
types.
 In-vivo delivery of naked DNA was first described in 1990.
 Efficiency of the delivery of naked DNA can be improved when
administered in a pressure-mediated fashion.
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 Particle bombardment technology enables the localized delivery of DNA

readily into skin or muscle.
 Another technique for delivery of naked DNA directly into target cells is
electroporation.
 The successful delivery of DNA by electroporation in-vivo has been
reported in tissues such as skin and muscle.
 In polymeric delivery systems, cationic polymers are used in gene delivery
because they can easily complex with the anionic DNA molecules.
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 The mechanism of action of these polycomplexes is based on the generation

of a positively charged complex owing to electrostatic interaction of these
cationic polymers with anionic DNA.
 Commonly used polymers include Polyethylenimine , Poly-L-
Lysine,Chitosans and Dendrimers.
 Agents such as Folates, Transferrin, Antibodies, or Sugars such as
Galactose and Mannose can be incorporated for tissue targeting. Synthetic
polymers such as Protective Interactive Noncondensing Polymers (PINC),
poly-L-lysine, cationic.
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 Polymers, and dendrimers offer an alternative to cationic lipids as a vehicle

for DNA delivery into target cells.
 Encapsulation of a DNA molecule or even a therapeutic viral vector within
a biodegradable polymer has been demonstrated to permit the controlled
release of the DNA in a targeted cell over a period of weeks or months.
 The inclusion of proteins and peptides in the DNA complex, which are
recognized by receptors on targeted cells, has led to an improvement in the
efficiency of DNA uptake in several instances.
 Some polymers have inherent potent pharmacological properties (such as
hypercholesterolemia induced by chitosans) that make them extremely
unfavorable for human use.
22 LIPOSOMAL GENE DELIVERY SYSTEMS

 Liposomes are one of the most versatile tools for the delivery of DNA
therapeutics.
 Liposome and drug/lipid complexes have been used for the delivery of the
anticancer drugs Doxorubicin and Daunorubicin.
 Liposomes can be used as DNA drug delivery systems either by entrapping
the DNA-based therapeutics inside the aqueous core or complexing them to
the phospholipids lamellae.
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 Liposome can also be used for specialized gene delivery options, such as
long circulation half-life and sustained and targeted delivery.
 Numerous studies have demonstrated the use of cationic liposomal
formulations for the delivery of different plasmid constructs in a wide range
of cells, both in-vivo and in-vitro.
 The use of cationic lipids to transfer DNA into cells was first described as
an in-vitro method of DNA delivery.
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 Cationic liposomes have also been used in clinical trials to deliver

therapeutic DNA.
 Cationic liposomal formulations consist of mixtures of cationic and
zwitterionic lipids.
 Proprietary formulations of cationic lipids such as Lipofectamine ,Effectene
and Tranfectam are commercially available, but most of the kits are useful
only for in-vitro experimentation.
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 There are reports of improved efficiency of DNA delivery by cationic lipid

via the coupling of specific receptor ligands or peptides to DNA/ liposome
complexes.
 Cytotoxicity of cationic lipids has been established in numerous in vitro and
in vivo studies.
 Low transfection efficiencies have been attributed to the heterogeneity and
instability of cationic lipoplexes.
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 Another drawback in the use of cationic lipids is their rapid inactivation in

the presence of serum.
 Some in-vivo studies have revealed that the gene transduction responses
obtained by cationic liposomes were transient and short-lived.
 As an alternative to cationic lipids, the potential of anionic lipids for DNA
delivery has been investigated.
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 The safety of anionic lipids has been demonstrated when administered to

epithelial lung tissue. In recent years, a few studies using anionic liposomal
DNA delivery vectors have been reported.
 There have been attempts to incorporate anionic liposomes into polymeric
delivery systems.
 However, these vectors have limited applications, mainly because of
Inefficient entrapment of DNA molecules within anionic liposomes
Lack of toxicity data.
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 Lack of further progress of these systems may be attributed, in part, to the

poor association between DNA molecules and anionic lipids, caused by
electrostatic repulsion between these negatively charged species.
 Along with numerous cationic and anionic lipid derivatives, functionalized
liposomal formulations serving specific therapeutic objectives have shown
promise in gene therapy.
 Specialized liposomal delivery platforms include pH-sensitive
liposomes,immunoliposomes, and stealth liposomes.
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 pH-sensitive liposomes can be generated by the inclusion of 1,2-dioleoyl-3-

phosphoethanolamine (DOPE) into liposomes composed of acidic lipids
such as cholesterylhemisuccinate or oleic acid.
 At the neutral cellular pH 7, these lipids have the typical bilayer structure;
however, upon endosomal compartmentalization, they undergo protonation
and collapse into a nonbilayer structure, thereby leading to the disruption
and destabilization of the endosomal bilayer, which in turn helps in the
rapid release of DNA into the cytoplasm.
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 Efficient gene delivery of the betagalactosidase and luciferase reporter

plasmids has been obtained using pH-sensitive liposomes in a variety of
mammalian cell lines.
 A chemical derivative of DOPE, cCitraconyl-DOPE, has been used to
deliver DNAbased therapeutics to cancer cells, thereby combining the
targeting and the rapid endosome-releasing aspects of specialized liposomal
delivery systems.
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 A phosphatidylcholine/glycyrrhizin combination was also successful in pH-

sensitive gene delivery in mice.
 Immunoliposomes are sophisticated gene delivery systems that can be used
for cell targeting by the incorporation of functionalized antibodies attached
to lipid bilayers.
 Immunoliposomes containing an antibody fragment against the human
transferrin receptor were successfully used in targeted delivery of
tumorsuppressing genes into tumors in-vivo .
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 Tissue-specific gene delivery using immunoliposomes has been achieved in

the brain, embryonic tissue and breast cancer tissue.
 Stealth liposomes are sterically stabilized liposomal formulations that
include polyethylene glycol (PEG)-conjugated lipids.
 Recently, polyethylenimine (PEI), poly(lactic-co-glycolic acid) (PLGA),
polypeptides, chitosan, cyclodextrin, dendrimers, and polymers containing
different nanoparticles are used in vitro and in vivo with respect to their
structure, physicochemical properties, and delivery efficiency as an siRNA
delivery vehicle.
33 REFERENCES

 NUCLEIC ACID AS THERAPEUTICS BY Saraswat Pushpendra,Pareek

Aravind & Bhandari Anil (PAGE NO:19 to 36)
 THANK YOU
34

FOR YOURS
PATIENCE 