Assignment on Recombinant DNA Technology and Gene Therapy

DNA= Deoxyribu-Nucelic Acid
• DNA is a very large molecule,
made up of smaller units called
nucleotides
• Each nucleotide has three parts: a
sugar (ribose), a phosphate
molecule, and a nitrogenous base.
• The nitrogenous base is the part of
the nucleotide that carries genetic
information
• The bases found in DNA are four:
adenine, cytosine, guanine, and
thymine ( ATP, CTP, GTP, and
TTP)
What is DNA?

• Recombinant DNA technology procedures
by which DNA from different species can
be isolated, cut and spliced together -- new
"recombinant " molecules are then
multiplied in quantity in populations of
rapidly dividing cells (e.g. bacteria, yeast).
Recombinant DNA Technology

• In the early 1970s it became possible to
isolate a specific piece of DNA out of the
millions of base pairs in a typical genome.
recombinant dna technology

Recombinant DNA technology is based on a
number of important things:
 Bacteria contain extra chromosomal
molecules of DNA called plasmids which
are circular.

 Bacteria also produce enzymes called
restriction endonucleases that cut DNA
molecules at specific places into many smaller
fragments called restriction fragments.
 There are many different kinds of restriction
endonucleases

Restriction Enzymes and plasmid
• Sticky end and blunt end are the two
possible configurations resulting from the
breaking of double-stranded DNA

When RES acts at the center of symmetry, two
complementary strands of DNA are of equal
length, hence forms the blunt end.
T C A G A T C A GA
A G T C T A G T CT

• Some RES breaks the DNA on either side of
center of symmetry with the liberation of
unequal fragments which are called as stick
ends/ cohesive ends.
• G A A T T C G A A T T C
• C T T A A G C T T A A G

Digestion of DNA by EcoRI to produce cohesive
ends.

• Restriction Enzymes are primarily found in
bacteria and are given abbreviations based
on genus and species of the bacteria.
• One of the first restriction enzymes to be
isolated was from EcoRI
• EcoRI is so named because it was isolated
from Escherichia coli strain called RY13.

• A gene is a stretch of DNA
that codes for a type of
protein that has a function
in the organism.
• It is a unit of heredity in a
living organism.. All living
things depend on genes
• Genes hold the information
to build and maintain an
organism's cells and pass
genetic traits to offspring.
What is gene?

• It can be defined as the isolation and
amplification of an individual gene sequence
by insertion of that individual gene sequence
into a bacterium where it can be replicated
Gene cloning

Step 1
A fragment of DNA,
containing the gene to
be cloned, is inserted
into a circular DNA
molecule called a
vector, to produce a
chimera or recombinant
DNA (rDNA)
molecule.
13
BASIC STEPS IN GENE CLONING

Step 2
The vector acts as a vehicle that transports the gene
into a host cell, which is usually a bacterium
although other types of living cell can be used. This
process is called transformation.

Step 3
Within the host cell the vector multiplies producing
numerous identical copies not only of itself but also
of the gene that it carries.

16
Step 4
When the host cell
divides, copies of
rDNA molecule are
passed to the
progeny and further
vector replication
takes place.

Step 5
After large no: of cell divisions a colony or
clone of identical host cells is produced. Each
cell in the clone contains one or more copies of
the rDNA molecule
Step 6
Then, the host cells are then lysed and rDNA
can be separated.

Recombinant DNA technology had made it possible to treat
different diseases by inserting new genes in place of
damaged and diseased genes in the human body.
Applications of rdna technology in medicine
Insulin is a hormone made up of protein. It is secreted in
the pancreas by some cells called as islet cells. If a
person has decreased amount of insulin in his body, he
will suffer from a disease called diabetes. Recombinant
DNA technology has allowed the scientists to develop
human insulin by using the bacteria as a host cell and it
is also available in the market. It is believed that the
drugs produced through microbes are safer.
Insulin:-

VACCINES:
Recombinant DNA technology enables the
scientists to develop vaccines by cloning the gene used
for protective antigen protein. Viral vaccines are most
commonly developed through this technology for
example, Herpes, Influenza, Hepatitis and Foot and
Mouth Diseases
Human Growth Hormones:-
In recent years, scientists have developed many growth
hormones using recombinant DNA technology. The
disease of dwarfism is treated with this hormone.

Infectious Diseases:-
Many diseases are diagnosed by conducting certain tests.
Recombinant DNA technology has allowed the development
of many tests which are being used to diagnose diseases like
TB and cancer.
In the diagnosis process, certain pathogens are isolated and
identified, and then diagnostic kits are produced when the
genome of the specific pathogen is known to kill it or block
its pathogenic activity.

PRODUCTION OF NOVEL PLANTS:
Rdna is used in distinguishing of novel agricultural plants
which are high yielding and pest resistant
Cloning of genes from wild pest resistant varieties has been
used.
Strain improvement for fermentation:
Rdna uses extensively for improvement of strains of
microbes.

REFERENCES:
 FUNDAMENTALS OF MEDICAL
BIOTECHNOLOGY: Author: Aparna Raja
Gopalan,editors: irfan ali khan, page no:203-226
 U.Sathyanarayana: biotechnology: page no: 530-542
 pharmaceutical biotechnology: fundamentals and
applications: Author: s s kori. Page no:74-80.

By
KAPIL
YADAV
M.PHARM
PHARMACOL
OGY

Overview
Definition.•
Brief Description.•
Why named so?•
Nomenclature.•
Characteristics.•
Mode of Actions.•
Types.•
Impacts & Uses.•
Use of Restriction Enzymes in•
Recombinant DNA Technonoly.
Restriction Enzyme Recognition•
Sequences.
Summary.•

RESTRICTION
ENZYME/RESTRICTION
ENDONUCLEASEEnzymes• that cut DNA at or near specific recognition
nucleotide sequences known as restriction sites.
• Especial class of enzymes that cleave (cut) DNA at a specific
unique internal location along its length.
• Often called restriction endonucleases (Because they cut
within the molecule).
Discovered• in the late 1970s by Werner Arber, Hamilton Smith,
and Daniel Nathans.
Essential• tools for recombinant DNA technology.
Naturally• produced by bacteria that use them as a defense
mechanism against viral infection.
• Chop up the viral nucleic acids and protect a bacterial cell by
hydrolyzing phage DNA.

RESTRICTIO
N ENZYME
• The bacterial DNA is protected from
digestion because the cell methylates
(adds methyl groups to) some of the
cytosines in its DNA.
• The purified forms of these bacterial
enzymes are used in today's laboratories.
• Commonly classified into three types,
which differ in their structure and
whether they cut their DNA substrate at
their recognition site, or if the
recognition and cleavage sites are
separate from one another.
• To cut DNA, all restriction enzymes make
two incisions, once through each sugar-
phosphate backbone (i.e. each strand) of
the DNA double helix.

Nomenclatur
e• Since their discovery in the
1970s, many restriction
enzymes have been
identified; for example,
more than 3500 different
Type II restriction enzymes
have been characterized.
• Each enzyme is named
after the bacterium from
which it was isolated,
using a naming system
based on bacterial genus,
species and strain.
• For example, the name of
the EcoRI restriction
enzyme was derived as
shown in the box.
Deriva tion of the EcoRI
na me
Abbreviation Meaning Description
E Escherichia genus
co coli specific epithet
R RY13 strain
I
First
identified
order of
identification
in the bacterium

Mode of action (how R.E.
cuts DNA)
The• enzyme makes two incisions, one through each of the sugar-
phosphate backbones (i.e., each strand) of the double helix
without damaging the nitrogenous bases.
Restriction• enzymes hydrolyze the backbone of DNA between
deoxyribose and phosphate groups. This leaves a phosphate
group on the 5' ends and a hydroxyl on the 3' ends of both
strands. A few restriction enzymes will cleave single stranded
DNA, although usually at low efficiency.
The• restriction enzymes most used in molecular biology labs
cut within their recognition sites and generate one of three
different types of ends. In the diagrams below, the recognition
site is boxed in yellow and the cut sites indicated by red
triangles.
5' overhangs: The enzyme cuts asymmetrically within the
recognition site such that a short single-stranded segment
extends from the 5' ends. BamHI cuts in this manner.

Mode of action (how R.E.
cuts DNA)
Blunts : Enzymes that cut at precisely opposite sites in the two
strands of DNA generate blunt ends without overhangs. Smai
is an example of an enzyme that generates blunt ends.
The 5' or 3' overhangs generated by enzymes that cut asymmetrically
are called sticky ends or cohesive ends, because they will readily stick
or anneal with their partner by base pairing. The sticky end is also called a
3' overhangs: Again, we see asymmetrical cutting within the
recognition site, but the result is a single-stranded overhang
from the two 3' ends. KpnI cuts in this manner.

Star Activity of
Restriction EnzymesStar activity is defined as the alteration in the digestion specificity that
occurs under sub-optimal enzyme conditions. Star activity results in
cleavage of DNA at non-specific sites. Some of the sub-optimal conditions
that result in star activity are as follows:
• pH >8.0
• glycerol concentration of >5%
• enzyme concentration >100 units/mg of DNA
• increased incubation time with the enzyme
• presence of organic solvents in the reaction mixture

Artificial restriction
enzymes Artificial restriction enzymes can be generated by
fusing a natural or engineered DNA binding domain
to a nuclease domain (often the cleavage domain of
the type IIS restriction enzyme FokI).
 Such artificial restriction enzymes can target large
DNA sites (up to 36 bp) and can be engineered to
bind to desired DNA sequences.
 Zinc finger nucleases - are the most commonly used
artificial restriction enzymes and are generally used
in genetic engineering applications, but can also be
used for more standard gene cloning applications.

RECOGNITION SEQUENCES AND CUTTING SITES FOR
SOME ENZYMES:

Restriction Enzyme
Recognition Sequences
• The length of restriction recognition sites
varies: The enzymes EcoRI, SacI and SstI each
recognize a 6 base-pair (bp) sequence of DNA,
whereas NotI recognizes a sequence 8 bp in
length, and the recognition site for Sau3AI is only
4 bp in length. Length of the recognition
sequence dictates how frequently the enzyme
will cut in a random sequence of DNA. Enzymes
with a 6 bp recognition site will cut, on average,
every 46 or 4096 bp; a 4 bp recognition site will
occur roughly every 256 bp.

Isoschizomer:
• These are pairs of restriction enzymes specific to the
same recognition sequence.
• For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are
isoschizomers of each other.
• The first enzyme discovered which recognizes a given
sequence is known as the prototype
e.g.
SphI CUTS ----------C G T A C | G--------
BbuI CUTS ----------C G T A C | G--------
e.g.
MboI CUTS ----------| G A T C --------
Sau3AI CUTS ----------| G A T C --------

Neoschizomers:
• An enzyme that recognizes the same sequence but cuts it
differently e.g, Smal (CCC/GGG) and XmaI (C/CCGGG) are
neoschizomers of each other. AatII (recognition sequence:
GACGT↓C) and ZraI (recognition sequence: GAC↓GTC) are
neoschizomers of one another
e.g.
SmaI CUTS ----------C C C | G G G--------
XmaI CUTS --------C | C C G G G--------

Isocaudomers:
• An enzyme that recognizes a slightly different sequence,
but produces the same ends. i.e blunt ends
e.g.
BamHI CUTS ----------G | G A T C C--------
BclI CUTS ----------T | G A T C A--------

Restriction Enzyme
Recognition Sequences
• Restriction recognitions sites can be unambiguous or ambiguous: The
enzyme BamHI recognizes the sequence GGATCC and no others - this is
what is meant by unambiguous. In contrast, HinfI recognizes a 5 bp
sequence starting with GA, ending in TC, and having any base between
(in the table, "N" stands for any nucleotide) - HinfI has an ambiguous
recognition site. XhoII also has an ambiguous recognition site: Py stands
for pyrimidine (T or C) and Pu for purine (A or G), so XhoII will recognize
and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC.
• Other point to notice from the table above is that most recognition
sequences are palindromes - they read the same forward (5' to 3' on
the top strand) and backward (5' to 3' on the bottom strand). Most, but
certainly not all recognition sites for commonly-used restriction
enzymes are palindromes.

THE IMPACT OF RESTRICTION ENZYMES
Genetic engineering
Type• II enzymes yielded many practical benefits, as E.
K12, its genes and its vectors became the workhorses of
molecular biology in the 1970s for cloning, generation of
libraries, DNA sequencing, detection and overproduction
of enzymes, hormones, etc.Production of insulin from recombinant bacteria and yeast by
Gene technology, thus greatly increasing the supply for diabetics
and the production of a recombinant vaccine for Hepatitis B by
Biogen to treat the hundreds of millions of people at risk of
infection by this virus

THE IMPACT OF RESTRICTION ENZYMES
DNA fingerprinting
• DNA fingerprinting allows the solution of paternity
cases, the identification of criminals and their victims
and the exoneration of the falsely accused. The use of
REases in this system enabled the creation of suitable
procedures for such identification.
• Useful for identifying pathogenic bacterial strains, most
recently of S. aureus sp with antibiotic-resistance and
virulence factors mediated by mobile genetic elements,
e.g. the methicillin-resistant S. aureus (MRSA) bacteria.

Why restriction enzymes
are important for rDNA
techniques?
• Restriction enzyme recognizes and cuts, or digests, only
one particular sequence of nucleotide bases in DNA
• Typical restriction enzymes used in cloning experiments
recognize four-, six-, or eight-base sequences
• It cuts this sequence in the same way each time..
• Hundreds of restriction enzymes are known, each
producing DNA fragments with characteristic ends.

GENE CLONING:
DNA fragments from different species
can be ligated to create Recombinant
DNA

The role of a
restriction
enzyme in
making
recombinant
DNA

RFLP:
Restriction Fragment Length Polymorphism is a tool
to study variations among individuals & among
species

DNA MAPPING:
Restriction Enzymes can be used
to generate a restriction map. This
can provide useful information in
characterizing a DNA molecule.

Reference
• https://en.wikipedia.org/wiki/Restriction_enzyme
• Lehninger’s Principles of Biochemistry
• http://www.biology-pages.info/R/RestrictionEnzymes.html
• https://www.neb.com/products/restriction-endonucleases/restriction-
endonucleases/types-of-restriction-endonucleases
• http://www.bio.miami.edu/dana/dox/restrictionenzymes.html
• http://www.bio.miami.edu/dana/dox/restrictionenzymes.html
• https://www.dnalc.org/resources/animations/restriction.html
• http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.h
tml
• http://www.biologydiscussion.com/dna/restriction-enzymes/restriction-
enzymes-in-dna-mode-of-action-and-its-types-2/12065
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874209/
• http://www.sigmaaldrich.com/technical-
documents/articles/biology/restriction-enzymes.html

By•
• KAPIL YADAV
M.PHARM
PHARMACOLOG•
Y

INTRODUCTION
A cloning vector is a small piece of DNA,
taken from a virus, a plasmid, or the cell of a
higher organism, that can be stably
maintained in an organism, and into which a
foreign DNA fragment can be inserted for
cloning purposes.

FEATURES OF A
CLONING VECTOR
 Origin of replication.
 Cloning site.
 Selectable marker.
 Reporter gene.

TYPES OF CLONING
VECTORS
1. Plasmid.
2. Bacteriophage.
3. Cosmid.
4. Bacterial Artificial Chromosome.
5. Yeast Artificial Chromosome.

PLASMID VECTOR
 Plasmid vector is a small,
piece of circular DNA
found outside of the
bacterial chromosome.
 Capable of
replication.
 Can transfer
autonomous
genes from
one cell to other.

 Contains an origin of replication, allowing for
replication independent of host’s genome.
 Contains Selective markers for the selection of
cells containing a plasmid.
 Contains a Multiple Cloning Site (MCS).
 Easy to be isolated from the host cell.
 E.g pBR322, pUC19.

Properties Of Plasmid Vectors
Sm aller plasmid vectors are preferred for many
reasons:
 The efficiency of transformation is inversely
related to the size of the plasmid.
Lar ger plasmids are more difficult to
characterize by restriction mapping.

 The yield of foreign DNA is reduced
with larger plasmids because these
plasmids
replicate to lower copy numbers

Varieties Of Plasmids Based
On Functions
 Fertility F-plasmids
which contain tra genes.
They are capable
of conjugation and
result in the expression
of sex pilli.

 Resistance (R) plasmids
which contain genes that
provide resistance against
antibiotics or poisons.

Col plasmids which contain genes that code
for bacteriocins, proteins that can kill other
bacteria.
 Degradative plasmids which enable the
e.g.digestion of unusual substances,
toluene and salicylic acid.
 Virulence plasmids which turn the
bacterium into a pathogen.

Uses Of Plasmid
 Major use of plasmids is to make large
amounts of proteins.
 Plasmid may also be used for gene transfer
into human cells as potential treatment in
gene therapy so that it may express the
protein that is lacking in the cells.

BACTERIOPHAGE VECTOR
 Cloning Vector that uses a Bacteriophage as a
means for making and storing exact copies of
segments of DNA.
 It infects bacteria.

TYPES
 The bacteriophages used for cloning are ;
i. phage λ
ii. M13 phage.

Phage λ Vector
 Infect E.coli.
 Origin of replication.
 Size is 48,502 bp.
 High transformation
efficiency, about 1000 times
more efficient than the
plasmid vector.
 Enterobacteria is important
in the study of specialized
transduction.

Types of Phage
λ Vector
a. Insertion vectors: contain a unique cleavage site
whereby foreign DNA with size of 5–11 kb may
be inserted. e.g λgt10 and λZAPII.
b. Replacement vectors: replacement vector has two
recognition sites for the restriction endonuclease
used for cloning. e.g λWES.λB' and λEMBL4

DNA Cloning Using Phages As
Vectors

Phage M13 Vector
 A gene for the lac repressor.
 The operator region of
the lac Z gene.
 A lac promoter upstream of
the lac Z gene.
 A polylinker region.

Types of M13 vector
 M13 mp1 form by introduction of the lacZ'
genes into the m13 vector.
 M13 mp2 has a slightly altered lacZ' gene.It is the
simplest M13 cloning vector.
 M13 mp7 form by the introduction of additional
restriction sites into the lacZ' gene. The polylinker is
inserted into the EcoRI site of M13mp2, to give
M13mp7.

 Complex M13 vectors have more complex
polylinkers inserted into the lacz' gene, ability to take
DNA fragments with two different sticky ends.
 M13mp9 having same polylinker as in m13 mp8 but
in the reverse orientation which is important in DNA
sequencing.

Use Of M13 Vector
 M13 vector use in nanostructures and
nanotechnology.

COSMID VECTOR
 Hybrids between a phage DNA molecule and
a bacterial plasmid.
 An origin of replication (ori).
 A cos site .
 An ampicillin resistance gene (amp).

 Restriction
cloning .
sites for
Cos mids can carry up to
50 kb of inserted DNA.

Uses Of Cosmids
 Cosmids can be used to build
libraries.
genomic
 They are often used as a cloning
vector in genetic engineering.

Bacterial Artificial
Chromosome
DN A construct, based on a functional F-plasmid.
 F-plasmids play a crucial role because they
contain partition genes that promote the even
distribution of plasmids after bacterial cell
division.
Ba cterial artificial chromosome's usual insert size
is 150-350 kbp.

 Like other vectors, BACs contain:
1. Origin (ori) sequence derived from an E. coli
plasmid called the F factor.
2. Multiple cloning sites (restriction sites).
3. Selectable markers (antibiotic resistance)

Uses Of BAC
 Useful for sequencing large stretches of
chromosomal DNA.
 Frequently
projects.
used in genome sequencing

YEAST ARTIFICIAL
CHROMOSOME
 YAC are genetically engineered chromosomes
derived from the DNA of the yeast.
 Capable of carrying inserts of 100 - 1000 kbp.
 A YAC can be considered as a functional
artificial chromosome since it includes three
specific DNA sequences:

 TEL: Telomere located at each chromosome
end, protects the linear DNA from degradation
by nucleases.
 CEN: Centromere which is the attachment site
for mitotic spindle fibers, "pulls" one copy of
each duplicated chromosome into each new
daughter cell.
 ORI: Replication origin sequences which are
specific DNA sequences.

 It also contains few other specific sequences like:
a. Selectable markers: that allow the easy
• isolation of yeast cells that have taken up the
artificial chromosome.
b. Recognition site: for the two restriction
• enzymes EcoRI and BamHI.

Uses Of YAC
 Used to express eukaryotic proteins that
require posttranslational modification.
Us ed for detailed mapping of specific
regions of the genome.

REFERENCES
• 1. Hall, RM; Collis, CM (1995). "Mobile gene cassettes and
integrons: Capture and spread of genes by site-specific
recombination". Molecular microbiology
• 2. Plant biotechnology – the genetic manipulatio by Adrian
Slater, Nigel W.Scott & Mark R. Fowlerns of plants 2nd edition
•
• 3. Principles of gene Manipulations & Genomics – Primrose
• 4. Joseph Sambrook, David Russell. "Chapter 1". Molecular
Cloning
• Laboratory Manual 1 (3rd ed.)
• 5. Lederberg, J and Lederberg, EM (1952) Replica plating and
indirect selection of bacterial mutants. J Bacteriol.

• Submitted To:
• DR. GOVIND SINGH
ASSISTANT PROFESSOR
• PHARMACOLOGY
• Submitted By:
• KAPIL YADAV
M.PHARM
PHARMACOLOGY•

• Revolutionized biology
• Manipulation of DNAsequences and the
construction of chimeric molecules, provides a
means of studying how a specific segment of
DNAworks
• Studies in bacteria and bacterial viruses have
led to methods to manipulate and recombine
DNA
• Once properly identified, the recombinant
DNAmolecules can be used in various ways
useful in medicine and human biology

Recombinant Pharmaceuticals
• Anumber of human disorders can be traced
to the absence or malfunction of a protein
normally synthesized in the body.
• Most of these disorders can be treated by
supplying the patient with the correct
version of the protein
• Hence, modern pharmaceutical
manufacturing frequently relies upon
recombinant drugs.

Recombinant Pharmaceuticals
Hu• man Insulin
Human• Growth Hormone
Human• blood clotting factors
• Vaccines
Mono• clonal Antibodies
Interfe• rons
Anti• biotics & other secondary metabolites

Human Insulin
Earl• iest use of recombinant technology
Modify• E.coli cells to produce insulin;
performed by Genentech in 1978
Prio• r, bovine and porcine insulin used but
induced immunogenic reactions
Also, the• re were many purification and
contamination hassles.
• Toovercometheseproblems,researchersinserted
human insulin genes into a suitable vector
(E.coli)

• Insulin is a hormone made up of protein. It is secreted in the
pancreas by some cells called as islet cells. If a person has
decreased amount of insulin in his body, he will suffer from a
disease called diabetes. Recombinant DNA technology has
allowed the scientists to develop human insulin by using the
bacteria as a host cell and it is also available in the market. It
is believed that the drugs produced through microbes are
safer.
Applications of rdna technology in medicine
Recombinant DNA technology had made it possible to
treat different diseases by inserting new genes in place
of damaged and diseased genes in the human body.

Producing Recombinant Insulin
• First, scientists synthesized genes for the two
insulinA& B chains.
• They were then inserted into plasmids along
with a strong lacZ promoter.
• The genes were inserted in such a way that the
insulin & B-galactosidase residues would be
separated by a methionine residue. This is so
that the insulinA& B chains can be separated
easily by adding cyanogen bromide.

Producing Recombinant Insulin
The• vector was then transformed into E.coli
cells.
Once• inside the bacteria, the genes were
"switched-on" by the bacteria to translate
the code into either the "A" chain or the
"B" chain proteins found in insulin
The• purified insulin A and B chains were
then attached to each other by disulphide
bond formation under laboratory conditions

Human Growth Hormones
Soma• tostatin and Somatotrophin are two
proteins that act in conjunction to control
growth processes in the human body, their
malfunction leading to painful and disabling
disorders such as Acromegaly (uncontrolled
bone growth) and Dwarfism.
Somatos• tatin was the first human protein to be
synthesized in E. coli. Being a very short
protein, only 14 amino acids in length, it was
ideally suited for artificial gene synthesis.

Production of Recombinant Human
Growth Hormones
• The strategy used was the same as described
for recombinant insulin, involving insertion
of the artificial gene into a lacZ′ vector,
synthesis of a fusion protein, and cleavage
with cyanogen bromide

Recombinant Blood Clotting
Factors
• Human factor VIII is a protein that plays a
central role in blood clotting.
• The commonest form of haemophilia in
humans results from an inability to synthesize
factor VIII
• The factor VIII gene is very large. The mRNA
codes for a large polypeptide (2351 amino
acids), which undergoes a complex series of
post-translational processing events, eventually
resulting in a dimeric protein consisting of a
large subunit and a small subunit.

Blood Clotting Factors
The• two subunits contain a total of 17
disulphide bonds and a number of glycosylated
sites. As might be anticipated for such a large
and complex protein, it has not been possible to
synthesize an active version in E. coli.
• TwoseparatefragmentsfromthecDNAwereused.
Each cDNA fragment was ligated into an
expression vector along with Ag promoter (a
hybrid between the chicken b-actin and rabbit
b-globin sequences) and a polyadenylation
signal from SV40 virus.
The pla• smid was introduced into a hamster cell
line and recombinant protein obtained.

Blood Clotting Factors
Alter• native method- pharming
The comp• lete human cDNA has been attached
to the promoter for the whey acidic protein
gene of pig, leading to synthesis of human
factor VIII in pig mammary tissue and
subsequent secretion of the protein in the milk.
The• factor VIII produced in this way appears
to be exactly the same as the native protein

Recombinant Vaccines
• Two types:
(i)Recombinant protein vaccines: This is based
on production of recombinant DNAwhich is
expressed to release the specific protein used in
vaccine preparation
(ii)DNAvaccines: Here the gene encoding for
immunogenic protein is isolated and used to
produce recombinant DNAwhich acts as
vaccine to be injected into the individual.

Recombinant protein vaccines:
• Apathogen produces its proteins in the body
which elicit an immune response from the
infected body.
• The gene encoding such a protein is isolated
from the causative organism
• This DNAis expressed in another host
organism, like genetically engineered
microbes; animal cells; plant cells; insect
larvae etc, resulting in the release of
appropriate proteins.
• These when injected into the body, causes
immunogenic response against the
corresponding disease providing immunity.

DNAvaccines:
Refers to• the recombinant vaccines in which the
DNA is used as a vaccine.
The• gene responsible for the immunogenic protein is
identified, isolated and cloned with corresponding
expression vector.
Upon int• roduction into the individuals to be
immunized, it produces a recombinant DNA.
This• DNA when expressed triggers an immune
response and the person becomes successfully
vaccinated.
The• mode of delivery of DNA vaccines include:
direct injection into muscle; use of vectors like
adenovirus, retrovirus etc; invitro transfer of the
gene into autologous cells and reimplantation of the
same and particle gun delivery of the DNA.

DNA vaccines:
In• certain cases, the responsible gene is
integrated into live vectors which are
introduced into individuals as vaccines.
This• is known as live recombinant vaccines. Eg:
vaccinia virus. Live vaccinia virus vaccine (VV
vaccine) with genes corresponding to several
diseases, when introduced into the body elicit
an immune response but does not actually
cause the diseases.

Recombinant Antibodies
• An immunoglobulin which produced because of the
introduction of an antigen into the body, and which
possesses the ability to recognize the antigen.
• Using recombinant antibody has significant
advantages compared with the conventional
antibody and there for its use becoming more
popular now days.
• The fact that no animals are needed in the
manufacturing procedure of the recombinant
antibodies, in addition, the manufacturing time is
relatively short compared with the conventional
method.
• Moreover, the quality of the final product is higher

Production of Recombinant
Antibodies
• The production of non-animal recombinant
antibodies can be broken down into five steps:
(1)creation of an antibody gene library
(2) display of the library on phage coats or cell
surfaces
(3)isolation of antibodies against an antigen of
interest
(4) modification of the isolated antibodies and
(5)scaled up production of selected antibodies in
a cell culture expression system.

Interferons
Interfe• rons (IFNs) are a group of signalling
proteins made and released by host cells in
response to the presence of pathogens, such
as viruses, bacteria, parasites,
or tumor cells.
In a typical• scenario, a virus-infected cell
will release interferons causing nearby cells
to heighten their anti-viral defenses.

Interferons
Recomb• inant DNA technology has proved the
most satisfactory route to the large scale
production of human interferons.
The g• enes of all three types of HuIFN have
been cloned in micro-organisms and expression
obtained.
Hu• IFNȕ and Ȗproduced in this manner lack
the glycosylation present in the naturally
occurring substances but this does not affect
their specific activity.

Interferons
• Greatly improved methods of purification,
including immuno-adsorption
chromatography on monoclonal antibody
columns, are now available so there should
be no difficulty in supplying adequate
amounts of very pure interferon of all three
types although, up till now, only HuIFNα
has been readily available.

Recombinant Secondary
Metabolites
• The importance of antibiotics to medicine has
led to much research into their discovery and
production.
• GM micro-organisms are used to increase
• production.
• Another technique used to increase yields is
gene amplification, where copies of genes
• coding for enzymes involved in the antibiotic
• production can be inserted back into a cell, via
vectors such as plasmids.

Production of Recombinant Plant
Secondary Metabolites
Plant• secondary metabolites can also be produced
by rDNA technology in plant suspension cultures,
micro-organism cultures and hairy root cultures
• A. rhizogenes mediated transformation which can
transfer foreign genes into the transformed hairy
root.
• E.g.: 6-hydroxylase gene of Hyoscyamus
muticus which was introduced to Atropa
belladonna using A. rhizogenes.
Eng• ineered roots showed an increased amount of
enzyme activity and a five-fold higher concentration
of scopolamine.

REFERENCE
• 1. Hall, RM; Collis, CM (1995). "Mobile gene cassettes and integrons: Capture
and spread of genes by site-specific recombination". Molecular microbiology
• 2. Plant biotechnology – the genetic manipulatio by Adrian Slater, Nigel
W.Scott & Mark R. Fowlerns of plants 2nd edition
•
• 3. Principles of gene Manipulations & Genomics – Primrose
• 4. Joseph Sambrook, David Russell. "Chapter 1". Molecular Cloning
Lab• oratory Manual 1 (3rd ed.)
• 5. Lederberg, J and Lederberg, EM (1952) Replica plating and indirect
selection of bacterial mutants. J Bacteriol.

GENE THERAPY-VARIOUS TYPES OF
GENE TRANSFER TECHNIQUES
PRESENTED BY:-KAPIL
YADAV

GENE TRANSFER
• Gene transfer is defined simply as a technique to stably
introduce foreign genes into the genome of target cells.
• The directed desirable gene transfer from one organism
to another and the subsequent stable integration &
expression of foreign gene into the genome is referred
as genetic transformation.
• Transient transformation occur when DNA is not
integreted
into host genome

• Stable transformation occur when DNA is integrated
into host genome and is inherited in subsequent
generations.
• The transferred gene is known as transgene and the
organism that develop after a successful gene transfer
is known as transgenic.

METHODS OF GENE
TRANSFER
DNA transfer by natural
methods
• 1. Conjugation
• 2. Bacterial transformation
• 3. Retroviral transduction

DNA TRANSFER BYARTIFICIAL
METHODS
• Physical methods
• 1. Microinjection
• 2. Biolistics transformation
• Chemical methods
• 1. DNA transfer by calcium phosphate
method
• 2. Liposome mediated transfer
• Electrical methods
• 1. Electroporation

CONJUGATION
• Requires the presence of a special plasmid
called the F plasmid.
• Bacteria that have a F plasmid are referred to as as
F+ or
male. Those that do not have an F plasmid are F- of
female.
• The F plasmid consists of 25 genes that mostly code
for
production of sex pilli.
• A conjugation event occurs when the male cell
extends his sex pilli and one attaches to the female.

• This attached pilus is a temporary cytoplasmic
bridge through which a replicating F plasmid is
transferred from the male to the female.
• When transfer is complete, the result is two male
cells.
• When the F+ plasmid is integrated within the
bacterial chromosome, the cell is called an Hfr cell
(high frequency of recombination cell).

TRANSFORMATION
Transformation is the direct uptake of exogenous DNA
from its
surroundings and taken up through the cell membrane .
Transforma• tion occurs naturally in some species of
bacteria, but it can also be effected by artificial
treatment in other species.
Cel• ls that have undergone this treatment are said to be
competent.
Any DNA th• at is not integrated into the chromosome
will be
degraded.

TRANSDUCTION
Gene• transfer from a donor to a recipient by way of a
bacteriophag..
If• the lysogenic cycle is adopted, the phage
chromosome is integrated (by covalent bonds) into the
bacterial chromosome, where it can remain dormant for
thousands of generation
The• lytic cycle leads to the production of new phage
particles
which are released by lysis of the host.

VECTORLESS or DIRECT
GENE
TRANSFER• Physical
methods
• 1.
Microinjection
•
•
•
•
•
•
•
2. Biolistics transformation
Chemical methods
1. DNA transfer by calcium phosphate
method
2. Liposome mediated transfer
3. Transfer of DNA by use of polyethene
glycol
Electrical methods
1. Electroporation

Electroporation
• Electroporation uses electrical pulse to produce
transient pores in the plasma membrane thereby
allowing DNA into the cells.
• These pores are known as electropores.
•

The c• ells are placed in a solution containing
DNA and subjected to electrical pulse to cause
holes in the membrane.
The fore• ign DNA fragments enter through holes
into the cytoplasm and then to nucleus.

Advantages of
Electroporation
• 1. Method is fast.
• 2. Less costly.
• 3. Applied for a number of
cell
types.
• 4. Simultaneously a large
number of cell can be treated.
• 5. High percentage of stable
transformants can be
produced

Microinjection
The microinjection is the process of transferring the
desirable DNA into the living cell ,through the use
of glass micropipette .
Glass micropipette is usually of 0.5 to 5
micrometer, easily penetrates into the cell
membrane and nuclear envelope.
The desired gene is then injected into the sub
cellular compartment and needle is removed

Limitations of
microinjection
• Costly.
• Skilled personal required.
• More useful for animal
cells.

Biolistics or
Microprojectiles
Biol• istics or particle bombardment is a
physical method that uses accelerated
microprojectiles to deliver DNA or other
molecules into intact tissues and cells.
The gene gun is a device that litera• lly fires
DNA into target cells .
The DNA to be• transformed into the cells is
coated onto
• microscopic beads made of either gold or
tungsten.

• The coated beads are then attached to the end of the
plastic bullet and loaded into the firing chamber of
the gene gun.
• An explosive force fires the bullet with DNA coated
beads towards the target cells that lie just beyond the
end of the barrel.
• Some of the beads pass through the cell wall into the
cytoplasm of the target cells

Liposome mediated gene
transfer
• Liposomes are spheres of lipids which can be used to
transport
molecules into the cells.
• These are artificial vesicles that can act as delivery
agents for
exogenous materials including transgenes.
• Promote transport after fusing with the cell membrane.
• Cationic lipids are those having a positive charge are
used for the transfer of nucleic acid.

Advantag
es
• 1. Simplicity.
• 2. Long term
stability.
• 3. Low toxicity.
• 4. Protection
of nucleic acid
from
degradation

Calcium phosphate mediated DNA
transfer
• The process of transfection involves the admixture of
isolated DNA (10-100ug) with solution of calcium
chloride and potassium phosphate so precipitate of
calcium phosphate to be formed.
• Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
• A fraction of cells will take up the calcium phosphate
DNA
precipitate by endocytosis.

Transfe• ction efficiencies is quite
low.

Polyethylene glycol mediated
transfectionThis• method is utilized for protoplast only.
Polyethylene glycol stimul• ates endocytosis and
therefore DNA
uptake occurs.
Pr• otoplasts are kept in the solution containing
polyethylene
glycol (PEG).
After transfer• of DNA to the protoplast in presence
of PEG and other chemicals, PEG is allowed to get
removed

• Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992).
Electroporation
• and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J.,
63 (5):
• 1320-1327.
• [2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian
cells with
• DNA. Nucleic Acids Res., 15 (3): 1311-1326.
• [3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M.,
Matsuo, N., Darnell,
• R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote
• neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci.
USA., 96
(17):
• 9885-9890.
• [4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation.
Methods., 24
• Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of
tissue USPTO Application #: 20080214986 - Class: 604 21 (USPTO)".
• Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced
membrane pores electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-
4622(84)87003-9. PMID 6722274.
• ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN

• Jogdand, S.N. (2006). Gene Biotechnology. Himalaya Publishing House. Mumbai, India. 2nd ed., p 237-
249.
• Chen, C.A. & Okayama, H. (1988). Calcium phosphate-mediated gene transfer: a highly efficient
trasfection system for stably transforming cells with plasmid DNA. Biotechniques.,
• Watwe, R.M. & Bellare, J.R. (1995). Manufacture of liposomes a review. Curr. Sci. India., . Nicolau, C.,
Legrand,
A. & Grosse, E. (1987). Liposomes as carriers for in vivo gene transfer and expression. Method
Enzymol., 149:
157-176.
• lies, M.A. & Balaban, A.T. (2001). Recent developments in cationic lipid-mediated gene delivery and
gene therapy.
Expert Opin. Ther. Patents.,
• Reece, R.J. (2004). Analysis of Genes and Genomes. John Wiley and Sons Ltd.
• Johnston, S.A. & Tang, D.C. (1994). Gene gun transfection of animal cells and genetic immunization.
Methods Cell
Biol., 43
Klein, T.M., Arentzen, R., Lewis, P.A. & Fitzpatrickmcelligott, S. (1992). Transformation of microbes, plants
and animals by particle bombardment. Biotechnology
• King, R. (2004). Gene delivery to mammalian cells by microinjection. Methods Mol. Biol.,
• David B. Burr; Matthew R. Allen (11 June 2013). Basic and Applied Bone Biology. Academic. p. 157. ISBN 978-
0-12-391459-0.
Retrieved 15 July 2013.
• ^ Juan Carlos Lacal; Rosario Perona; James Feramisco (11 June 1999). Microinjection. Springer. p. 9. ISBN
•

APPLICATIONS AND RECENT
ADVANCES OF GENE THERAPY
• PRESENTED BY:
• Kapil Yadav
• M.PHARM 1st YEAR
• Pharmacology
PRESENTED TO:•
Dr. Govind•
Singh
Assistant•
Professor

WHAT IS GENE THERAPY
Gene therapy is an experimental technique that uses genes to•
treat or prevent disease.
In the future, this technique may allow doctors to treat a•
disorder by inserting a gene into a patient cells instead of using
drugs or surgery.
Gene therapy was first conceptualized in• 1972.
Researchers are testing several approaches to gene therapy,•
including:
 Replacing a mutated gene that causes disease with a healthy
copy of a gene.
 Inactivating, or “knocking out” a mutated gene that is
functioning improperly.
 Introducing a new gene into the body to help fight a disease.

APPLICATIONS OF GENE THERAPY
 Gene therapy is used for the treatment of disorder
involving blood cells can be cured by this method.
The hematopoietic stem cells are removed from an
affected individual and transfected with functional
genes. The engineered stem cells are then re-
injected into the individual.
This method is used with individual suffering from
SCID resulting from a defective gene encoding
adenosine diaminase (ADA).

1. THERAPY FOR CYSTIC FIBROSIS
Cystic fibrosis is a fatal genetic disease
characterized by accumulation of sticky
,dehydrated mucous in respiratory tract and
lungs
In the patient of Cystic fibrosis the CFTR (Cystic
fibrosis transmembrane regulator) protein is not
produced due to gene defect.
The chloride ion concentrate within the cell
which draw water from surrounding.
As a result respiratory tract and lungs becomes
dehydrated with sticky mucous.

CONTINUED…
 In Gene therapy, adenoviral vector system have been used.
 Adenovirus do not integrate themselves into host cells.
 By using adeno-associated virus vector system, some good
results were reported in the gene therapy of cystic fibrosis.

2. THERAPY FOR SCID (ADA DEFICIENCY)
Severe Combined Immunodeficiency Disease•
This is inherited immune disorder associated with T -
lymphocytes, and B-lymphocytes dysfunction.
Deficiency of ADA accumulate and destroy T -lymphocytes.
 T-lymphocytes are essential for body ̍s immunity.
Patient of SCID (lacking ADA) die at an young age.

TREATMENT
• A plasmid vector bearing a proviral DNA is
selected.
• A part of proviral DNA is replaced by ADA gene.
• Circulating lymphocytes are removed.
• The cells are transfected with ADA gene.
• The genetically-modified lymphocytes are

Continued…
• Infuse lymphocytes with ADA gene.
• Expressed into patient.
• Synthesis of ADA in child with SCID
• Correction of SCID

3. Therapy for Lesch-Nyhan syndrome
• The most likely candidates for future gene
therapy trials will be rare diseases such as
Lesch-Nyhan syndrome, a distressing
disease in which the patients are unable to
manufacture a particular enzyme. This leads
to a bizarre impulse for self- mutilation,
including very severe biting of the lips and
fingers. The normal version of the defective
gene in this disease has now been cloned.

X-linked recessive Disease
LNS• is transmitted as and X-
linked recessive trait. Female
carriers do not show the symptoms.
LNS is characterized by self-
mutilating behaviours such as lip
and finger biting and/or head
banging. The deficiency of HGPRT
activity leads to accumulation of
guanine• .
By using retroviral vector•
system, HGPRT producing genes
were inserted into cultured
human bone marrow cells.

4. THERAPY FOR HEMOPHILIA
• Hemophilia is a genetic disease due to lack of a
gene that encodes for clotting factor IX.
• It is characterized by excessive bleeding.
• By using a retroviral system ,genes for the
synthesis of factor IX were inserted into the liver
cells.

5. THERAPY FOR BLINDNESS(LCA)
•  Leber's congenital amaurosis (LCA) is a rare
• inherited eye disease that appears at birth or in
• the first few months of life.
•  Characterized by no pupillary responses and
• severe vision loss and blindness.
•  Researchers at Moorfields Eye Hospital and
• University College London in London conducted
• the first gene therapy clinical trial for patients
• with RPE65 LCA.

CHM-(CHOROIDEREMIA)
 Choroideremia is caused by a loss-of-function mutation in
the CHM gene which encodes Rab escort protein 1 (REP1), a
protein involved in lipid modification of Rab proteins
 While the complete mechanism of disease is not fully
understood, the lack of a functional protein in the retina
results in cell death and the gradual deterioration of
the choroid, retinal pigment epithelium (RPE), and
retinal photoreceptor cells.
 As of 2017, there is no treatment for
choroideremia; however, retinal gene
therapy clinical trials have demonstrated a

6. THERAPY FOR CANCER
• � Multiple gene therapy strategies have been
developed to treat a wide variety of cancers,•
including suicide gene therapy, anti• -
angiogenesis
and therapeutic gene vaccines.•
• � Two-thirds of all gene therapy trials are for
cancer and many of these are entering the•
advanced stage e.g. gene vaccine trials for•
prostate cancer and pancreas cancer.•

7. THERAPY FOR HIV
•  FDA Approves Further Study Of Promising Gene
• Therapy HIV Treatment (19 March, 2015).
•  Experimental stem cell gene therapy that could act as
• functional cure for HIV infection has been approved by
• the FDA to move into early human test trials.
•  Cells harvested from a positive person’s body. The stem
• cells are genetically manipulated to develop into white
• blood cells that are missing the key cellular receptors
• that the HIV virus uses to insert its genetic code into
• healthy cells. The modification effectively models a HIV positive
• person’s white blood cells .

8. THERAPY FOR GAUCHER DISEASE
 Gaucher disease is a lysosomal storage
disease which is characterized by deficient
activity of lysosomal enzyme, known as
glucocerebrosidase. This resulted in
progressive accumulation of glucocerebroside
only in bone marrow.

The extensive study based on transferring
therapeutic gene to hematopoietic stem cell.

RECENT ADVANCES IN
GENE THERAPY

The Beginning…
• � In the 1980s, Scientists began to
look into
gene therapy.•
• � They would insert human genes
into a bacteria cell.
• � Then the bacteria cell would
transcribe and translate the
information into a protein
• � Then they would introduce the
protein into human cells

• On September 14, 1990 at the U.S. National Institutes of Health, W. French
• Anderson M.D. and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and
Kenneth Culver, M.D., performed the first approved gene therapy procedure on
four-year old Ashanthi DeSilva, Born with a rare genetic disease called severe
combined immunodeficiency (SCID).
• What did they do
• In Ashanthi's gene therapy procedure, doctors removed white blood cells from the
• child's body, let the cells grow in the laboratory, inserted the missing gene into the
• cells, and then infused the genetically modified blood cells back into the patient's
• bloodstream.
•
First Approved Gene Therapy

Current Status
 FDA hasn’t approved any human gene therapy product for sale.
Reasons:
 In 1999, 18-year-old Jesse Gelsinger died from multiple organ
failure 4 days after treatment for ornithine transcarboxylase
deficiency.
• .
 Death was triggered by severe immune response to adenovirus
carrier.
 January 2003, halt to using retrovirus vectors in blood stem cells
because children developed leukemia-like condition after
successful treatment for X-linked severe combined
immunodeficiency disease.

RECENT ADVANCES IN GENE THERAPY
•  Although early clinical failures led many to dismiss gene therapy as
• over-hyped, clinical successes since 2006 have bolstered new
optimism
• in the promise of gene therapy.
•  These include successful treatment of patients with the retinal
disease, X-linked SCID, chronic lymphocytic leukemia(CLL),
acute lymphocytic leukemia(ALL), multiple myeloma and Parkinson's
disease.
•
•  These recent clinical successes have led to a renewed interest in gene
• therapy, with several articles in scientific and popular publications
• calling for continued investment in the field.

 More than 5000 patients
have been treated in last ~12
years worldwide.

A success story
• As of early 2007, she was still in good health, and
• she was attending college. Some would state that the
• study is of great importance despite its indefinite
• results, if only because it demonstrated that gene
• therapy could be practically attempted without
• adverse consequences

Cindy Kisik and Ashanthi in 1992 with the pioneer
physicians of gene therapy: (from left) French Anderson,
MD; Michael Blaese, MD; and Kenneth Culver.

R. Michael Blaese, MD with Ashanthi DeSilva (left)
and Cindy Kisik at the IDF 2013 National Conference,
June 29

'mending broken hearts' by using gene
therapy
Novel• techniques to “mend broken hearts” using gene therapy and stem cells
represent a major new frontier in the treatment of heart disease•
• � It was achieved by the researchers at Gladstone Institute of Cardiovascular Disease in
California•
• � They were able to re-programme scar-forming cells into heart muscle cells, some of
which were capable of transmitting the kind of electrical signals that make the heart beat•
• � They performed on a live mice, transforming scar-forming cells, called fibroblasts,
into beating heart muscle cells•
• � They injected three genes (cocktail of genes) into the heart of live mice that had been
damaged by heart attack, fibroblasts could be turned into working heart cells.•
• � Researchers said that the “cocktail of genes” used to regenerate cells could one day be
replaced with• “small drug-like molecules” that would offer safer and easier delivery

First Real-Time MRI-Guided Gene Therapy for Brain
Cancer
• �Neurosurgeons at the University of California, San Diego School of Medicine and UC San
Diego• Moores Cancer Center are among the first in the world to utilize real-time magnetic
resonance imaging (MRI) guidance for delivery of gene therapy as a potential treatment for•
brain tumors•
• �Using MRI navigational technology, neurosurgeons can inject Toca 511 (vocimagene
amiretrorepvec• ), a novel investigational gene therapy, directly into a brain malignancy
• �The new approach offers a precise way to deliver a therapeutic virus designed to make the
tumor susceptible to cancer• -killing drugs

Continued…
• Toca 511 is a retrovirus engineered to
• selectively replicate in cancer cells, such as
• glioblastomas.
• Toca 511 produces an enzyme that
converts
• an anti-fungal drug, flucytosine (5-FC), into
• the anti-cancer drug 5-fluorouracil (5-FU).
• After the injection of Toca 511, the
patients
• are treated with an investigational extended
release
• oral formulation of 5-FC called Toca
• FC.
• Cancer cell killing takes place when 5-FC
• comes into contact with cells infected with
• Toca 511.

stem cell gene therapy gives hope to
prevent inherited neurological disease
• Scientists from The University of Manchester have used stem cell gene
• therapy to treat a fatal genetic brain disease
•  It was used to treat Sanfilippo – a fatal inherited condition which causes
• progressive dementia in children
•  Sanfilippo, is currently untreatable mucopolysaccharide (MPS) disease
•  It is caused by the lack of SGSH enzyme in the body which helps to breakdown
• and recycle long chain sugars, such as heparan sulphate (HS)
•  Children with the condition build up and store excess HS throughout their body
• from birth which affects their brain and results in progressive dementia and
• hyperactivity, followed by losing the ability to walk and swallow

Continued…
• � Researchers have developed a stem cell gene therapy which overproduces
the SGSH enzyme specifically in bone marrow white blood cells to increase•
SGSH enzyme from bone marrow transplants, and to target it to the cells that•
traffic into the brain•
• � It was seen that mice treated by this method produce five times the normal
SGSH enzyme levels in the bone marrow and• and 11 per cent of normal levels
in the brain•
• � The enzyme is taken up by affected brain cells and is enough to correct
brain HS storage and• neuro inflammation to near normal levels and
completely corrects the hyperactive• behaviour in mice with Sanfilippo

Mucopolysaccharidosis Type IIIA
potential gene therapy
• � Mucopolysaccharidosis Type IIIA (MPSIIIA) is a metabolic disorder in which the body
is missing an enzyme that is required to break down long chains of sugars known as•
glycosaminoglycans•
• � The glycosaminoglycans collect in the body and cause damage, particularly in the
brain if not broken•
• � Fàtima Bosch and colleagues at Universitat Autònoma de Barcelona in Spain
developed a form of gene therapy to replace the enzyme that is missing in MPSIIIA•
• � They injected the replacement gene into the cerebrospinal fluid that surrounds the
brain
and spinal cord•
• � This study demonstrates that gene therapy can be delivered to the brain through the
cerebrospinal fluid and suggests that this approach could potentially be used as a•
therapy
for MPSIIIA•

IS GENE THERAPY TOTALLY SAFE ??
•  Although gene therapy is a promising treatment
• option for a number of diseases (including inherited
• disorders, some types of cancer, and certain viral
• infections), the technique remains risky and is still
• under study to make sure that it will be safe and
• effective.
•  Gene therapy is currently only being tested for the
• treatment of diseases that have no other cures

Technical Difficulties in Gene
Therapy
•  Gene delivery: Successful gene delivery is not easy or
• predictable, even in single-gene disorders.
•  For example, although the genetic basis of cystic fibrosis is
well
• known, the presence of mucus in the lungs makes it
physically
• difficult to deliver genes to the target lung cells.
•  Delivery of genes for cancer therapy may also be
complicated
• by the disease being present at several sites.
•  Gene-therapy trials for X-linked severe combined
• immunodeficiency (X-SCID), however, have been more
• successful

Problems with Gene Therapy
Short Lived•
Hard to rapidly integrate therapeutic DNA into genome and rapidly•
dividing nature
of cells prevent gene therapy from long time•
Would have to have multiple rounds of therapy•
Immune Response•
new things introduced leads to immune response•
increased response when a repeat offender enters•
the gene might be over• -expressed (toxicity)
Viral Vectors•
patient could have toxic, immune, inflammatory response•
also may cause disease once inside•

Continued…
Multigene• Disorders
Heart disease, high blood pressure, Alzheimer• ’s,
arthritis and diabetes are hard to
treat because you need to introduce more than one•
gene

Updates on current advances in gene
therapy
• At present, the three main
gene therapy strategies for
treatment of cancer are
application to oncolytic viruses,
suicide-gene therapy and gene-
based immunotherapy.

AGGRESSIVE GENE THERAPY FOR CANCER
• When dealing with cancer we need to destroy
the cancer cells, or at least inhibit their growth
and division.
• Several strategies have been used and may be
classified as follows:-
(a) Genetic replacement
(b) Direct attack
(c) Suicide
(d) Immune provocation

(A) Gene replacement
Gene replacement therapy for cancer is use in
correcting hereditary defect
The cancer is analyzed to identify the mutant
gene(s) that are responsible.
Oncogene or tumour suppression gene is then
inserted into the cancer cells.
For example, p 53 gene has been delivered to p53
deficient cancer cells.
The delivery method is usually via an adenovirus
vector, but sometimes liposomes have been used.

(B) Direct attack
In the direct plan of attack, a gene that helps kill cancer cells is used. For
example, the TNF gene encodes tumour necrosis factor.
This is produced by white blood cells known as tumor infiltrating
lymphocytes.
The cells normally infiltrate into tumour where they release TNF, which is
fairly effective at eradicating small cancers.
To attack a large cancer that is out of control, TNF production must be
increased. First the TNF gene is cloned. Then white blood cells are
removed from the patient and cultured.
Multiple copies of the TNF gene -or an improved TNF gene with enhanced
activity-are introduced into white cells.
Then the white cells are injected back into patient.
•

(C) SUICIDE GENE THERAPY
 Suicide strategy is a hybrid of anticancer drug therapy with gene transfer
therapy.
• Suicide gene therapy begins by delivering a therapeutic gene into the cancer
cells.
 The genes encodes an enzyme that will convert a nontoxic product into a
toxic compound.
 Because non-cancerous cells do not have the suicide enzyme, they are not
affected.
 The non-toxic prodrug, ganciclovir, is converted to its monophosphate by
thymidine kinase.
 Because only the cancer cells have thymidine kinase, all the noncancerous
cells are unaffected.
 Normal cellular enzyme then convert monophosphate to GCV-TP. This act as
a DNA chain terminator.
 DNA synthesis is inhibited and the cell is killed.

(D) IMMUNE PROVOCATION
 A more indirect approach relies on the body’s natural defences.
 Our immune systems are effective at killing cancers, provided they
identify them while still small.
 To survive, a cancer has to somehow evade the body’s immune
survillance.
 In this approach, gene therapy inserts a gene that attract the attention
of the immune system to the tumor cells.
 A related approach is to use cytokines. These are short proteins that
attract immune cells and stimulate their division and development.
 The genes for several cytokines of the interleukin family (especially IL2,
IL4 and IL12) have been used to provoke immune attack on cancer cells.
•

SUCCESS CASES OF GENE
THERAPHY

GENE THERAPY REDUCES
PARKINSON’S DISEASE SYMPTOMS
• � It significantly improved the weakness of the
• symptoms such as tremors, motor skill problems,
and
• rigidity.
• � Main- overactive brain region: the subthalamic
• nucleus should be introduced with gene.
• � That would produce GABA—an inhibitory
• chemical—then they could potentially quiet that
• brain region and alleviate tremors.

HOW IT WORKS ??
•  Done with local anesthesia, used a
harmless,
• inactive virus [AAV-2 GAD].
•  Deliver the GAD gene into patient’s
• subthalamic nucleus.
•  The gene instructs cells to begin making
• GABA neurotransmitters to re-establish
the
• normal chemical balance that becomes
• dysfunctional as the disease progresses.

REFERENCES
• Jogdand, S.N. (2006). Gene Biotechnology. Himalaya Publishing House. Mumbai, India. 2nd ed., p 237-249.
• Chen, C.A. & Okayama, H. (1988). Calcium phosphate-mediated gene transfer: a highly efficient
trasfection system for stably transforming cells with plasmid DNA. Biotechniques.,
• Watwe, R.M. & Bellare, J.R. (1995). Manufacture of liposomes a review. Curr. Sci. India., . Nicolau, C., Legrand,
A.& Grosse, E. (1987). Liposomes as carriers for in vivo
gene transfer and expression. Method Enzymol., 149:
• 157-176.
• lies, M.A. & Balaban, A.T. (2001). Recent developments in cationic lipid-mediated gene delivery and gene
therapy.
• Expert Opin. Ther. Patents.,
• Reece, R.J. (2004). Analysis of Genes and Genomes. John Wiley and Sons Ltd.
• Johnston, S.A. & Tang, D.C. (1994). Gene gun transfection of animal cells and genetic immunization. Methods
Cell
• Biol., 43
•Klein, T.M., Arentzen, R., Lewis, P.A. & Fitzpatrickmcelligott, S. (1992). Transformation of microbes, plants and
animals by particle bombardment. Biotechnology
• King, R. (2004). Gene delivery to mammalian cells by microinjection. Methods Mol. Biol.,
• David B. Burr; Matthew R. Allen (11 June 2013). Basic and Applied Bone Biology. Academic. p. 157. ISBN 978-0-12-
391459-0.
• Retrieved 15 July 2013.
• ^ Juan Carlos Lacal; Rosario Perona; James Feramisco (11 June 1999). Microinjection. Springer. p. 9. ISBN 978-3-7643-
6019-1. Retrieved 13 July 2013.

Assignment on Recombinant DNA Technology and Gene Therapy

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