Gene therapy is a form of molecular medicine that has the potential to influence significantly human health in this 21st century. It promises to provide new treatments for a large number of inherited and acquired diseases (Verma and Weitzman, 2005). The basic concept of gene therapy is simple which includes introduction of a piece of genetic material into target cells that will result in either a cure for the disease or a slowdown in the progression of the disease. To achieve this goal, gene therapy requires technologies capable of gene transfer into a wide variety of cells, tissues, and organs. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery.

1. Nawkar Ganesh Mahadeo

2. Key Interactions Between Fields of Biology and
Nanotechnology
Model
Biology Nanotechnology
Tools

3. DNA / Gene Therapy
 DNA / Gene therapy is defined as the transfer of genetic material into
a cell for therapeutic benefit
 A "correct copy" or "wild type" gene is inserted into the genome
 The most common type of vectors are viruses
 Target cells such as the patient's liver or lung cells are infected with
the vector
 It promises to provide new treatments for a large number of inherited
and acquired diseases
(Verma and Weitzman, 2005)

4. DNA Vaccines
 DNA vaccines consist of a DNA molecule, generally a circular
plasmid, with a gene that codes for the protein against which an
immune response is desired
 The first demonstration of a plasmid-induced immune response was
when mice inoculated with a plasmid expressing human growth
hormone elicited antibodies instead of altering growth
(Tang et al., 1992)
 They are capable of providing a broad, long lasting immune response
 They are relatively simple, cheap and quick to produce and they are
stable at room temperature

5. DNA Vaccination vs Gene Therapy
 DNA Vaccines  Gene Therapy
• The purpose of influencing the • The purpose of carrying out a
immune system specific function
• It aims to produce large • It aimed at achieving a long lasting,
amounts of protein in a short physiologically matched expression
span of time so as to generate of the gene, without activating the
an immune response immune system
• Does not requires a more • It requires advanced technologies
targeted and finely tuned to target gene at specific site and
technology its expression
• Aimed at a short-term presence • Aimed at presence of the added
of DNA in the animal is the genetic material over a longer
desired result period of time
• E.g. vaccines for HIV, herpes, • E.g. Single gene defect disorders,
hepatitis and influenza cancer etc

6. Types of DNA / Gene Therapy
 Germ line DNA / Gene therapy
• Germ cells - sperm or eggs, are modified by the introduction of
functional genes
• Results in heritable change
• Prohibited for application in human beings
 Somatic cell DNA / Gene therapy
• The gene is introduced only in somatic cells
• Expression of the introduced gene relieves/ eliminates symptoms of
the disorder
• Effect is not heritable
• Somatic cell therapy is the only feasible option

7. Genetic Diseases Potential Candidates for Gene Therapy
Defective gene Disease
1. Adenosine deaminase Severe Combined Immunodeficiency
2. Cystic fibrosis transmembrane regulator Cystic fibrosis
3. Factor IX Hemophilia B
4. Factor VIII Hemophilia A
5. Glucocerebrosidase Gaucher’s Disease
6. Low-density lipoprotein receptor Familial Hypercholesterolemia
7. 3-Globin Sickle Cell Anemia
 Three of the genetic diseases listed in table are presently the subject of gene
therapy clinical trials
• Adenosine Deaminase deficiency using T lymphocytes,
• Familial Hypercholesterolemia using hepatocytes, and
• Hemophilia using fibroblasts

8. Different Methods of Gene Delivery
Viral gene transfer Non-viral gene transfer
1. RNA virus vectors 1. Electroporation
e.g. Oncoretroviruses, 2. Microinjection
Lentiviruses, 3. Naked DNA
Spumaviruses 4. Particle Bombardment
5. Ultrasound
2. DNA virus vectors
Novel gene transfer
e. g. Adenoviruses,
Adeno-Associated Nanoparticles
viruses, Herpesvirus e.g. Liposomes, Gold
Nanoparticles, Magnetic
Nanoparticles

9. Viral Vector Construction
( Verma and Weitzman, 2005)

10. First Approved Gene Therapy Procedure
 Ashanthi De Silva - A rare genetic disease called
severe combined immunodeficiency (SCID)
 Defective adenosine deaminase gene results in
deficiency of ADA protein
 It plays important role in deamination reaction
Deoxyadenosine ADA Deoxyinosine
Dr. W. French Anderson
with four-year old
 Causes toxicity of T lymphocytes
Ashanthi De Silva at U.S.
National Institutes of
Health  Lack of healthy immune system

11. Gene Therapy Strategy
 Isolated T lymphocytes from patient and cultured in laboratory conditions
 The correct copy of ADA gene was introduced into the T-cells using a
retroviral vector
 Following transduction, the cells ware grown in culture to attained
significant number of cells
 Gene engineered cells given back to the patient in procedure similar to a
blood transfusion
 The amount of the ADA protein in the T-cells has risen to 25% normal

12. Why ADA Deficiency was First Target of Gene
Therapy?
 The ADA gene had been cloned earlier
 The gene is of average size and can easily be inserted into a
retroviral vector
 Bone marrow transplantation vs T cell replacement
 The amount of the ADA protein that needs to be produced in
order to maintain a functioning immune system is only 5-10 % of
normal

13. Limitations of Viral Mediated DNA delivery
 Toxicity and immunogenicity
 Restricted targeting of specific cell types
 Limited DNA carrying capacity e.g. for rAAV, commonly reported as 4.7kb
(Flotte, 2000)
 Production and packaging problems
 Recombination and random integration into host genome
 High cost

14. Failures of Viral Mediated Gene Therapy
 Retroviral vector
• Dr. Alan Fischer – Conducting gene therapy on SCID-X1 linked hereditary
disorder
• Hematopoietic stem cells from patients were stimulated and transduced ex
vivo with MLV-based retroviral vector
• Expressing the γc cytokine receptor subunit, and then were reinfused into
the patients
• During a 10-month follow up, γ c-expressing T and NK cells counts and
function were comparable to age-matched controls
• Two of the children developed T-cell leukemia
(Cavazzana et al., 2000)

15. Contd…
 Adeno-Associated Virus Vector
• Patients suffering from hemophilia B were treated with AAV vectors
expressing human factor IX
• Intramuscular injecting AAV factor IX vectors directly into liver, which in
turn have shown some unexplained toxicity
 University of Pennsylvania (1999)
• A human Phase I clinical trial for ornithine transcarbamylase deficiencies
• This trial was designed to test the safety of an E1/E4- deleted recombinant
adenovirus vector
• Jessie Gelsinger received highest dose and first person to die as result of
vector delivery ( Raper et al., 2003)

16. Nanobiotechnology
 Nanobiotechnology is a rapidly advancing area of scientific and
technological opportunity that applies the tools and processes of nano/
microfabrication to build devices for studying biosystems
Fig. Nanobiotechnology Interdisiplinary
Integration
 Applications of nanobiotechnology are in various fields such as
predictive diagnosis, medical care, drug discovery and environment

17. What is Nanoscale?
 “Nano” means dwarf in Greek
 Nanocsale : 1 nm = 1 x 10-9 m
Water Nanodevices White Tennis ball
molecule blood cell
Nanopores
Dendrimers
Nanotubes
Quantum dots
Nanoshells

18. The Timeframe of Nanobiotechnology

19. Applications of Different Nanoparticles in Medicine
 Liposomes
• Liposomes are phospholipid vesicles (50–100 nm)
• They have a bilayer membrane structure similar to that of
biological membranes and an internal aqueous phase
• Liposomes show excellent circulation, penetration and
Liposomes diffusion properties
 Dendrimers
• These are highly branched synthetic polymers (<15 nm)
• It show layered architectures constituted of a central
core, an internal region and numerous terminal groups
• Wide application in Drug Delivery System (DDS) and
Dendrimers gene delivery

20. Contd…
 Carbon nanotubes
• These are formed of coaxial graphite sheets (<100 nm)
rolled up into cylinders
• It exhibit excellent strength and electrical properties and
are efficient heat conductors
• Due to semiconductor nature of nanotubes are used as
Carbon nanotubes biosensors
 Magnetic nanoparticles
• These are spherical nanocrystals of 10–20 nm of size
with a Fe2+ and Fe3+ core surrounded by dextran or PEG
molecules
• Their magnetic properties make them excellent agents
to label biomolecules in bioassays, as well as MRI
Magnetic contrast agents
nanoparticles • Useful in targeted gene delivery

21. Contd…
 Quantum dots
• These are colloidal fluorescent semiconductor
nanocrystals (2–10 nm)
• They are resistant to photobleaching and show
exceptional resistance to photo and chemical
degradation
• Quantum dots excellent contrast agents for
Quantum dots imaging and labels for bioassays
 Gold nanoparticles
• These are one type of metallic nanoparticle of
size <50 nm
• These are prepared with different geometries,
such as nanospheres, nanoshells, nanorods or
Gold
nanocages
nanoparticles • These are excellent labels for biosensors

22. Ideal Characteristics of NP Gene Vector System
 A safe and efficient NP gene vector system must fulfill the following
four requirements
1) Particle sizes must be in the submicron range that facilitates the
penetration of the NPs through the cellular membrane
2) The possibility of surface modification that permits binding of NPs
with the pDNA and enhances the stability of the NP-DNA complex
3) Biodegradability, so that the accumulated NPs in cells could be
degraded
4) High transfection efficiency

23. Hurdles in DNA Delivery
Fig. DNA delivery pathways with three major barriers
(A) DNA–complex formation (B) Uptake (C) Endocytosis (endosome)
(D) Escape from endosome (E) Degradation (edosome)
(F) Intracellular release (G) Degradation (cytosol)
(H) Nuclear targeting (I) Nuclear entry and expression

24. Polyion Complex (PIC) Micelles for Plasmid DNA
Delivery
Fig. Polymeric micelles as intelligent nanocarriers for drug and gene delivery

25. Development of Polyion Complex (PIC) Micelle
 Biocompatibility of the polyplexes improved by using PEG -b- polycation
copolymers which electrostatically interact with pDNA to protect DNA from
enzymatic and hydrolytic degradation
 pDNA /PEG -b-PLL micelles intravenously injected intact
pDNA observed in blood circulation after 3 hr
(Harada-Shiba et al., 2002)
 PIC micelles stabilized by disulfide cross linking
 The intravenous injection of cross linked PIC micelles into mice resulted in
a uniform gene expression in the liver
(Miyata et al., 2005)
 To achieve a site-specific gene delivery, polyplex micelles might be
modified with targetable ligands such as peptides and antibodies
(Merdan et al., 2003)

26. A-B-C type Triblock Copolymer
A) PEG Segment
B) poly[(3-morpholinopropyl) aspartamide] (PMPA) as a low pKa polycation
C) PLL segment
(Fukushima et al., 2005)

27. Dendritic Photosensitizer for Light-induced Gene
Transfer
 pDNA condensation = quadruplicated
cationic peptide (CP4) + nuclear
localization signal (NLS)
 Anionic DPc (Dendritic phthalocya-
nine) = photosensitizer
 Mechanism
Cellular uptake of the ternary complexes
via endocytosis,
Dissociation of DPc from the complexes
in acidic vesicles due to the protonation of
the carboxyl groups on the dendrimer
periphery
Endosomal escape of the pDNA/CP4
Fig. pDNA/CP4/DPc ternary complexes
complexes to the cytoplasm upon photo
irradiation (Nishiyama et al., 2005)

28. Advantages of Nanocarriers over Viral Vector
 They are easy to prepare and to scale-up
 They are more flexible with regard to the size of the DNA being transferred
e.g. DNA compacted nanoparticles can contain plasmids up to 20 kb
(Fink et al., 2006)
 They do not elicit a specific immune response and can therefore be
administered repeatedly
 They are better for delivering cytokine genes
 They show little to no toxicity in the targeted tissues, and modest immune
response when high concentration
(Cooper, 2007; Farjo et al., 2006)
 Targeted gene delivery is possible

29. Future Challenges in Nanoparticle Application
 It is not yet possible to predict nanoparticle biodistribution according
to their physicochemical properties
 Once nanoparticles reach their target site, and despite their small
size, they do not enter into biological systems, such as cells or
organelles, easily
 Inside the cell, nanoparticles can remain structurally unaltered, can
be modified or can be metabolized
 More study is required about toxic effects of nanoparticles

30. Case Study

31. Objectives
 To develop the preparation protocol of PBCA-CTAB NPs
 To study its characteristics
 To develop the AFP-positive hepatocellular carcinoma gene
therapy using the PBCA-CTAB NP–pAFP-TK complex
 To study the expression of pAFP-TK in vitro

32. Materials and Methods
 HepG2, HeLa, and 3T3 cells (American Type Culture Collection)
 Escherichia coli DH5 α and pAFP-TK plasmid
 Enhanced green fluorescent protein plasmid N1 (pEGFP-N1) from
Clonetech
 Herpes simplex virus thymidine kinase (HSV-TK) primers
 A-butyl-ester cyanoacrylic-acid (BCA) from Baiyun Limited Co.
 Cetyltrimethylammonium bromide (CTAB) from Sigma
 3-(4,5-dimethyl-2-thiazolyl)-2,5–diphenyl-2H- tetrazolium bromide
(MTT) and DNase I from Sigma
 Equipment used
• Zetasizer 3000 and AJ-III Atomic Force Microscope (AFM)

33. AFP Photograph
Contd…
 Amplification and purification of plasmid DNA
 Preparation of PBCA NPs
• PBCA NPs were prepared by an emulsion polymerization method
• Tween 80 was dissolved in distilled water (pH 2.8)
• PBCA was added in slowly and mixed by magnetic stirring at room
temperature (22 °C-25 °C) for 5 hours
• Centrifuged at room temperature at 5000 rpm for 15 minutes
• The suspension was filtered by polyethylene terephthalate nuclear membrane
filter (diameter of pores = 0.22 µm)

34. Contd…
 Surface modification of PBCA-CTAB NPs
1 hr incubation
• 16.6 ml of PBCA NP solution (0.625%, w/v) @ 4000 rpm
for 30 min
+ 33.33 ml of CTAB solution (0.25%, w/v)
• Precipitation washed with dd H2O and resuspended in 150 mM NaCl
• Mixture lyophilized to steady state
 Characterization of PBCA NPs and PBCA-CTAB NPs
• PBCA-CTAB NPs were uniform and that the average diameters were
between 80 and 200 nm
• Zeta potential of the NPs revealed a positive surface charge of +15.6 mV

35. Results
 Cell viability
• Cytotoxicity of PBCA NPs and
PBCA-CTAB NPs to HepG2 cells
and 3T3 cells was estimated by
MTT assay
• The toxicity of NPs would suddenly Cytotoxicity of PBCA NPs
strengthen with increasing
concentration
No. Cell Type Concentration of NPs
1. HepG2 100 ng/µl
2. 3T3 200 ng/µl
Cytotoxicity of CTAB PBCA NPs

36. DNA Loading Efficiency of PBCA-CTAB NPs
 The difference between the total
amount of pDNA added in the NP
preparation buffer and the amount
of non-entrapped pDNA remaining
in the aqueous suspension
 PBCA CTAB NP solution + pDNA
in 50-µL reaction system (pH = 7)
 Results in different kinds of PBCA
CTAB- pDNA NPs in which the ratio Fig. The change in DNA loading
of PBCA-CTAB NPs to pDNA was efficiency of various NPs
1:1, 5:1, 10:1, 15:1, 20:1, 30:1, and
50:1, respectively

37. Gel Retarding Analysis and Protection Effect of NPs to
pDNA
 PBCA-CTAB-pDNA complexes
(containing 2 µg pDNA) were 1:1 5:1 10:1 15:1 30:1 50:1 10:1
incubated with 2 µl of RNase-free
DNase I solution (1 µg/µl) in 50 µl
of reaction buffer for 15 minutes
at 37 ° C
 The reaction was stopped by Fig. Electrophoretic mobility
adding 2 µL of RQ1 DNase stop analyses of PBCA-CTAB NP–pDNA
solution complexes
 The integrity of the pDNA was
analyzed by gel electrophoresis
(1% agarose)

38. In vitro Gene Transfection Efficiency
 The transfection efficiency of
PBCA-CTAB NPs was evaluated in
HepG2 cells and 3T3 cells using
the enhanced green fluorescent
protein (EGFP) gene as a reporter
 Super- Fect Transfection Reagent
was used as a positive control
Fig. The expression of the EGFP gene
 Naked pDNA was used as negative loaded by
control A) PBCA-CTAB NPs expressed in HepG2
cells
B) SuperFect Transfection Reagent
 The results, observed by inversion expressed in HepG2 cells
fluorescence microscope after C) PBCA-CTAB NPs expressed in 3T3 cells
transfection D) SuperFect Transfection Reagent
expressed in 3T3 cells.

39. RT-PCR Analysis
 To detect the expression of the
HSV-TK gene
 Expression of β-actin mRNA
was detected as an internal
standard
Fig. Expression of the TK gene in three
 PBCA NPs modified with CTAB kinds of cells transfected by different
pDNAs
can enter the cells effectively
with exogenous genes, which *(pAFP-TK gene, p3.1-TK and pcDNA3.1)
can also normally express in
cells
 The expression of the TK gene in the AFP-positive cells was controlled by
AFP enhancer more strongly than cytomegalovirus

40. Sensitivity of Transfected Cells to GCV
 MTT assay was used to examine
the sensitivity of GCV to
transfected HepG2 cells
 The concentration of GCV was 10
µg/ml then the cell viability was not
influenced
 The concentration of GCV was 50
µg/ml then 50% of cells were killed Fig. Examination by MTT assay of
sensitivity to GCV of transfected HepG2
cells
 GCV had a dose-dependent effect
on survival of AFP-positive cells

41. Apoptosis Induced by PBCA-CTAB NP - Mediated
pAFP - TK/ GCV System
 HepG2 cells transfected by pAFP-
TK loaded by PBCA-CTAB NPs
 Stained using Hoechst 33258
stain
 After treatment of GCV, the
nucleus of cells was condensed Fig. Apoptosis of cells induced by GCV after
transfection by pAFP-TK plasmid
 It confirmed one of the C)Fluorescence staining of transfected
mechanisms of the lethal effect of HepG2 cells not treated by GCV
the PBCA-CTAB NP-mediated D)Fluorescence staining of transfected
pAFP-TK/GCV system HepG2 cells treated by GCV

42. Summary
 Developed a novel transfection vector, PBCA-CTAB NPs - a
non-viral vector that can deliver DNA into targeted cells
 The pAFP-TK/GCV suicide gene therapy system have a high
transfection efficiency in AFP-positive cells and potent
antitumoral activities in vitro
 The system may be an effective candidate vector for treatment
of AFP-positive tumors