GENETIC

ENGINEERING
&
GENE THERAPY
Outline
Genetic Engineering
What is it?
What it can do?
Methods
Case studies
Gene Silencing
Gene therapy

How to introduce a new character
into an organism?
Random mutation
~ By chemicals, radiation
~ Affects the DNA
easy & cheap to do in a large scale
No information about the biology required

Outcome not predictable
Limited outcome
Screening of mutants difficult
Reversion back to the wild state

Genetic Engineering
Deliberate insertion of gene encoding the
desired character into the organism
Outcome predictable (often)
Stable cell lines
Complex (wide) range of recombinants
Can be applied even to higher organisms
like mammals

Central Dogma
DNA RNA Protein

The genetic code is universal

Since genetic code is universal, common
machinery exists in all organisms for
producing the protein from DNA
The Basic Tools
1. DNA (and RNA)
2. Restriction Enzymes
3. Vector
4. Host (Expression System)

Simply.
1. Isolate DNA from organism (e.g., extract DNA)

2. Cut DNA with restriction enzymes to a desired size.

3. Splice (or ligate) each piece of DNA into a cloning vector to
create a recombinant DNA molecule.

Cloning vector = artificial DNA molecule capable of replicating in a
host organism (e.g., bacteria).

4. Transform recombinant DNA (cloning vector + DNA
fragment) into a host that will replicate and transfer copies to
progeny.

DNA basics
~ In the DNA double helix, purines and pyrimidines face each other

~The two polynucleotide chains in the double helix are connected
by hydrogen bonds between the bases

~Watson-Crick base-pairing rules
A = T
G C

~ GC base pairs (bps)have more energy than AT bps

~ Since one strand of DNA is complementary to the other, genetic
material can be accurately reproduced; each strand serves as the
template for the synthesis of the other
Hydrogen Bonds
H
H
H
H
O
O
H
C
C
C
C
N
N
C
Thymine
H
N
H
H
N
C
C
C
C
N
N
H
N
C
Adenine
H
O
N
H
C
C
C
N
N
C
Cytosine
H
H
H
N
C
C
C
C
N
N
H
N
C
Guanine
N
H
O
H
In DNA
Restriction Enzymes
Most restriction enzymes occur naturally in bacteria.

Protect bacteria against viruses by cutting up viral DNA.

More than 400 restriction enzymes have been isolated.

Names typically begin with 3 italicized letters.

Enzyme Source

EcoRI E. coli RY13
HindIII Haemophilus influenzae Rd
BamHI Bacillus amyloliquefaciens H

Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.

Bacteria protects their DNA by modifying possible restriction sites
(methylation).

Short restriction site sequences occur more frequently in the
genome than longer restriction site sequences, e.g., (1/4)
n
.

Creating a Recombinant DNA
Vectors
Six different types of cloning vectors:

1. Plasmid cloning vectors

2. Phage cloning vectors

3. Cosmid cloning vectors

4. Shuttle vectors

5. Yeast artificial chromosomes (YACs)

6. Bacterial artificial chromosomes (BACs)
Plasmid Cloning Vectors:

Bacterial plasmids, naturally occurring, circular double-stranded
extrachromosomal DNA elements capable of replicating
autonomously within a cell.

Plasmid vectors engineered from bacterial plasmids for use in
cloning.

Required features (e.g., E. coli plasmid vectors):

1. Origin sequence (ori) required for replication.

2. Selectable trait that enables E. coli that carry the plasmid to
be separated from E. coli that do not (e.g., antibiotic
resistance, grow cells on antibiotic; only those cells with the
anti-biotic resistance grow in colony).

3. Unique restriction site such that an enzyme cuts the plasmid
DNA only once. A fragment of DNA cut with the same
enzyme can then be inserted into the plasmid restriction site.

4. Simple marker that allows you to distinguish plasmids that
contain inserts from those that do not (e.g., lacZ+ gene)
Insertion of DNA into host
Several methods
1. Transformation
2. Virus (Phage)
3. Electroporation
4. Gene gun
5. Microinjection
6. Liposomes ??

After Transformation
A
C B
A : Cells with NO plasmids
B : Cells with non-recombinant (original) plasmids
C : Cells with recombinant plasmids (required cells)
In Summary
Blue-White selection of
Recombinants
Culture the transformants on a nutrient plate
containing antibiotic and X-gal
Only cells with plasmids (recombinant & non-
recominant) will grow since antibiotic resistance
absent in plasmid-free cells
Cells with non recombinant plasmid will produce
blue colonies (X-gal breakdown)
Cells with recombinant plasmid will produce
white colonies (no X-gal breakdown)


Case Study: The Use of
Recombinant DNA to
Produce Human Insulin

Why synthesize human insulin?
Patients immune systems do not produce
antibodies against human insulin as they
do with bovine or porcine insulin
Projected decline in the production of
animal-derived insulin
Need for a more reliable and sustainable
method of obtaining the product
Why is insulin needed?
Protein hormone produced by beta cells of
islets of Langerhans in the pancreas
Regulates blood sugar by allowing uptake
of glucose from bloodstream into body
cells
Patients with diabetes have insufficient or
impaired production of insulin
Structure of Insulin
Two polypeptide chains; one with 21
amino acids and the second with 30 amino
acids

Chains are linked via a disulfide bond

Gene encoding the insulin protein is found
on chromosome 11
insulin
Restriction
enzymes used to
cut out insulin
gene and to cut a
bacterial
(E. coli) plasmid at
the same sticky
ends

insulin
Mutant strains of E. coli used to avoid
bacteria attacking foreign genes
Insert insulin gene next to E. coli beta-
galactosidase gene which controls
transcription
Bacterial cells replicate and make copies of
insulin gene

Inclusion Bodies of
the over-expressed
proteins
insulin
Insulin protein is purified (B-galactosidase
removed)
Chains are mixed and disulfide bridges
form
Yeast cells provide a sterile growth
medium
Final product is Humulin - chemically
identical to human insulin
Other examples
Growth hormone
Follicle-stimulating hormone
Erythropoietin
L-Asparaginase
Tissue plasminogen activator
Interferons
Limitations of Bacterial Host
Protein folding not always satisfactory

Post-translational modifications not satisfactory

Endotoxins ?
Alternatives
Insect cell systems
Fish cell systems
Plant cell systems
Mammalian cell systems
Transgenic animals
GENE THERAPY
Genes
Are carried on a chromosome

Encode how to make a protein
DNARNA proteins

Proteins carry out most of lifes function.

When altered causes dysfunction of a protein

When there is a mutation in the gene, then it will change the codon, which
will change which amino acid is called for which will change the
conformation of the protein which will change the function of the protein.

Genetic disorders result from mutations in the genome.


What is Gene Therapy
It is a technique for correcting defective genes
that are responsible for disease development
There are four approaches:
1. A normal gene inserted to compensate for a
nonfunctional gene.
2. An abnormal gene traded for a normal gene
3. An abnormal gene repaired through selective reverse
mutation
4. Change the regulation of gene pairs

Candidates for gene therapy
How It Works?
Deliver the therapeutic gene (a piece of
DNA) into a patients target cell
The therapeutic gene incorporates into the
host genome
Functional proteins are created from the
therapeutic gene causing the cell to return
to a normal state

How to deliver the gene?
# Viruses
~ Replicate by inserting their DNA into a host cell
~ Gene therapy can use this to insert genes that
encode for a desired protein to create the
desired trait

~ Four different types
~ Retroviruses
~ Adenoviruses
~ Adeno-associated Viruses
~ Herpes Simplex Viruses
Non-viral Options
Direct introduction of therapeutic DNA
But only with certain tissue
Requires a lot of DNA
Creation of artificial lipid sphere with aqueous core,
liposome
Carries therapeutic DNA through membrane
Chemically linking DNA to molecule that will bind to
special cell receptors
DNA is engulfed by cell membrane
Less effective
Trying to introduce a 47th chromosome
Exist alongside the 46 others
Could carry a lot of information
But how to get the big molecule through membranes?
Success?
The first gene therapy
was performed on
September 14
th
, 1990
Ashanti DeSilva was
treated for SCID
Sever combined
immunodeficiency


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

Viral Vectors
patient could have toxic, immune, inflammatory response
Reversion to virulent form
Multigene Disorders
Heart disease, high blood pressure, Alzheimers, arthritis and diabetes are
hard to treat because you need to introduce more than one gene
May induce a tumor if integrated in a tumor suppressor gene
because insertional mutagenesis

Concerns
FDA hasnt 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 omithine
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

But
SCID
Parkinsons disease
Cystic fibrosis
Sickle Cell anemia (mouse model)
DNA vaccines
Cancer

GENE SILENCING
When some misfunctional genes are turned
on, they produce proteins which harm the
body
Example : some cancers, autoimmune
diseases

Solution:
Block the messenger RNA from being
translated to protein



Small interfering RNA (siRNA)
Bottle Necks
~Variability in genome
(Single Nucleotide Polymorphisms)

~Delivery of DNA and RNA

~ Ethical questions
THANK YOU!