LETHAL GENES

AND
GENE THERAPY
BY ANILA AND POOJA

LETHAL GENES
Genes which result in the
reduction of viability of an
individual or become a cause for
death of individuals carrying
them are called as lethal genes.
Certain genes are absolutely
essential for survival. Mutation in
these genes creates lethal allele.

Lethal genes were first

discovered
by
Lucien
Cunot while studying the
inheritance of coat colour in
mice.
He expected a phenotype
ratio from a cross of 3
yellow:1 white, but the
observed ratio was 2:1.

TYPES OF LETHAL ALLELES

Lethal alleles fall into four
categories.
1.Early onset- lethal alleles
which result in death of an
organism at early stage of
life, for example, during
embryogenesis.
2. Late onset- lethal allele
which kills organism at their

3. Conditional- lethal allele which kill

an
organism
under
certain
environmental conditions only.
e.g., some temperature sensitive
alleles kill organisms only at high
temperature.
4. Semi lethal Lethal allele which
kill only some individuals of the
population but not all are know as
semi lethal.

Lethal alleles are dominant or

recessive.
Fully dominant lethal allele kills
organism in both homozygous and
heterozygous condition.
Recessive lethal
organisms
in
condition only.

alleles kills
homozygous

DOMINANT LETHAL GENES

Dominant lethal allele kills both in
homozygous and heterozygous
states.
Individuals with a dominant lethal
allele die before they can leave
progeny.
Therefore, the mutant dominant
lethal is removed from the population
in the same generation in which it
arose.

EXAMPLES OF DOMINANT LETHAL

ALLELES
An example is the "creeper" allele in
chickens, which causes the legs to be
short and stunted.
Creeper is a dominant gene, heterozygous
chickens display the creeper phenotype.
If two creeper chickens are crossed, one
would expect to have (from Mendelian
genetics) 3/4 of the offspring to be creeper
and 1/4 to be normal.
Instead the ratio obtained is 2/3 creeper
and 1/3 normal.

HUNTINGTONS DISEASE
Huntington disease is a
progressive brain disorder that
causes uncontrolled movements,
emotional problems, and loss of
thinking ability (cognition).
Adult-onset Huntington disease,
the most common form of this
disorder, usually appears in a
person's thirties or forties.

 Mutations in theHTTgene cause

Huntington disease.
TheHTTgene provides
instructions for making a protein
called huntingtin.
HH Individual dies of
Huntingtons disease
Hh Individual dies of
Huntingtons disease
hh Normal individual

 Early signs and symptoms can

include irritability, depression, small
involuntary movements, poor
coordination, and trouble learning
new information or making decisions.
Affected individuals may have
trouble walking, speaking, and
swallowing.
Individuals with the adult-onset form
of Huntington disease usually live
about 15 to 20 years after signs and
symptoms begin.

RECESSIVE LETHAL GENES

Recessive lethal genes kill only
when they are in homozygous
state. They may be of two kinds:
one which has no obvious
phenotypic effect in
heterozygotes.
one which exhibits a distinctive
phenotype when heterozygous.

EXAMPLES OF RECESSIVE LETHAL

ALLELLES
Brachydactyly
A genetic state in which the fingers are
unusually short in heterozygotic condition.
But, this condition is lethal during early
years to homozygous recessive individuals
due to major skeletal defects.
Most surgeries for brachydactyly are
cosmetic.
Some therapy might be needed to help
with kinesthetic activities.

What causes Brachydactyly?

A mutation occurs in IHH gene which
encodes proteins responsible for bone
growth and differentiation.
When a single mutated copy of the allele is
present, the phenotype has just few
deformations of skeletal bones. This is
because one dose of functional IHH allele
is almost enough to produce a required
amount of a protein essential for a skeletal
formation.
If an organism inherits two mutated copies
of IHH allele no protein essential for
skeletal bones formation is produced and

Let's say that an alleleais recessive

and codes for a completely
dysfunctional form of a protein
essential for bone growth, andAis a
dominant wild type allele. If
heterozygotes for these alleles
procreate, then:

Sickle Cell Anemia

A genetic state that is often fatal in the
homozygous recessive condition.
People who inherit one good copy of the
gene and one mutated copy are carriers.
They are clinically normal, but can still
pass the defective gene to their children.
When sickle-shaped red blood cells get
stuck in blood vessels, patients can have
episodes of pain called crises. Other
symptoms include delayed growth,
strokes, and jaundice (yellowish skin and
eyes because of liver damage).

Genetics of Sickle Cell

Anemia
Genotypes
Phenotypes
HbNHbN

Normal haemoglobin

HbNHbS

Sickle cell trait

HbSHbS

Sickle cell anaemia

Animation showing sickle

cells

Cystic Fibrosis
A genetic state that is fatal to
every homozygous recessive
person by age 30.
Sticky mucus accumulates in the
lungs giving rise to constant and
risky respiratory infections.
It is caused due to malfunctioning
of chloride ion channels in ducts.

Lungs in cystic fibrosis

Normal lung

Normal alveolar appearance

CF lung

Dilated crypts
filled with mucus and
bacteria.

Congenital Ichthyosis
Children with this disease are born with
crusted leathery skin with deep splits.
These splits lead to bleeding, infection and
death.
In Ichthyosis, the skin's natural shedding
process is slowed or inhibited and in some
types, skin cells are produced too rapidly.
Most types of autosomal recessive
congenital ichthyosis require two forms of
treatment - a reduction in the amount of
scale buildup and moisturising of the
underlying skin.

COAT COLOUR IN MICE

The coat colour of mice is governed
by a multiple allelic series in which A
allele determines agouti, AY allele
determines yellow coat and a allele
forms black coat.
The dominance hierarchy is as
follows:
AY > A > a
The AY allele acts as recessive lethal,
since in the homozygous state (AY
Y

Thus, when two

yellow coated
heterozygotes
(AY A) are
crossed, they
produce a
progeny showing
a ratio of 2:1
since
homozygous
yellow
individuals (AY
AY) are never

GENE THERAPY

Gene therapy is the

insertion of genes into
individual cells and
tissues to treat a disease
in which a defective
mutant allele is replaced
with a functional one

There are four

approaches:
A normal gene inserted to
compensate for a non-functional
gene.
An abnormal gene expression is
suppressed.
An abnormal gene repaired
through selective reverse
mutation.

TYPES OF GENE THERAPY

SOMATIC CELL GENE
THERAPY
Therapeutic

genes
are transferred into
the somatic cells.
Eg: introduction of
genes into the
bone marrow cells,
skin cells, blood
cells etc
It is non heritable
and cannot be
passed on to the

GERM LINE GENE THERAPY

Therapeutic genes
are transferred into
the germ cells.
Eg : genes
introduced into the
eggs and sperms
It is heritable and
passed on to the
next generations.

SOMATIC CELL THERAPY

GERM LINE THERAPY

STEPS IN GENE THERAPY

Identification of the defective gene.
Cloning of normal healthy gene.
Identification of target cell / tissue / organ.
Insertion of the normal functional gene
into the host DNA
5. Transferred gene (TRANSGENE) encodes
& produces proteins
6. The Proteins encoded by Transgene
corrects the disorder
1.
2.
3.
4.

APPROACHES TO
GENE THERAPY
EXVIVO

IN-VIVO

EX-VIVO APPROACH
Target cells are
removed from the body
and grown in vitro.
The gene is then
introduced into the
cultured cells.
These cells are then reintroduced into the
same individual
Examples: Fibroblast
cells, Hematopoietic
cells.

EXAMPLE OF EX-VIVO GENE

THERAPY
1st gene therapy to correct deficiency of
enzyme, Adenosine deaminase (ADA).
Performed on a 4yr old girl Ashanti DeSilva.
Was suffering from SCID- Severe
Combined Immunodeficiency.
Caused due to defect in gene coding for ADA

STEPS:
1. Cloned
therapeutic gene
is introduced
directly into the
affected tissue,
without removing
cells from the
body.
2. Specially
designed vehicles
are needed.
3. Examples are:
Lungs, Brain

In vivo approach:
(Direct Gene
Transfer)

Video of gene therapy

EXAMPLE OF IN VIVO-GENE
THERAPY
In patients with cystic fibrosis, a protein called
cystic fibrosis trans-membrane regulator
(CFTR) is absent due to a gene defect.
In the absence of CFTR chloride ions
concentrate within the cells and it draws
water from surrounding.
This leads to the accumulation of sticky
mucous in respiratory tract and lungs.
Treated by in vivo replacement of defective
gene by adenovirus vector .

ROLE OF VECTORS IN GENE

THERAPY

METHODS OF GENE
DELIVERY

Viral Vectors:

Adenovirus
Retrovirus
Adeno-associated virus (AAV)
Herpes simplex virus (HSV)

Non-viral vector based:

Naked DNA (plasmid DNA):
injection or gene gun
Liposomes (cationic lipids): mix
with genes

VIRAL VECTORS

ADENOVIRAL VECTORS
They are double stranded DNA
genome.
Adenoviruses are able to deliver
large DNA particles (up to 38 kb).
They do not integrate into the
host genome, their gene
expression is too short term.

 Advantages
High titers
Both dividing and non-dividing cells
Wide tissue tropism
Easily modify tissue tropism

Disadvantages
Transient expression ( not good for
genetic diseases)
Highly immunogenic
High titers of virus can be toxic
More suitable for cancer immunotherapy

RETRO VIRAL VECTOR

The genetic material inretrovirusesis in the
form ofRNAmolecules.
They can transfect dividing cells because
they can pass through the nuclear pores of
mitotic cells.
Retroviruses are useful forex vivodelivery
of somatic cells because of their ability to
linearly integrate into host cell genome.
Double stranded DNA copies are produced
from RNA genome.
The retrovirus goes through reverse
transcription using reverse transcriptase
and RNA
the double stranded viral genome

CHARACTERISTICS OF
RETRO VIRUS
Advantages
Integration: permanent expression
Good for genetic disorders

Disadvantages
Only infecting dividing cells
Insertional mutagenesis

ADENO-ASSOCIATED VIRUS
VECTORS
It is a human virus that can integrate
into chromosome 19.
It is a single stranded, non pathogenic
small DNA virus.
AAV enters host cell, becomes double
stranded and gets integrated into
chromosome.

CHARACTERISTICS OF AAV
VECTOR
Advantages
Integration and persistent
expression
No insertional mutagenesis
Infecting dividing and nondividing
cells
Safe

Disadvantages
Size limitation, 4.9 kb
Low titer of virus, low level of gene

HERPES VIRAL VECTORS

The genome consists of one double-stranded DNA
molecule which is 120 to 200 kilobases in length.
The virus itself is transmitted by direct contact and
replicates in the skin or mucosal membranes before
infecting cells of the nervous system.
They allow for a large DNA insert of up to or greater
than 20 kilobases; they have an extremely high titer.
They have been shown to express transgenes for a
long period of time in the central nervous system.
They are far from complete and require much
additional engineering to be as efficient as hoped.

WHY USE VIRAL VECTORS?

They are obligate intracellular
parasites
Very efficient at transferring viral
DNA into host cells
Specific target cells: depending
on the viral attachment proteins
(capsid or glycoproteins)
Gene replacement: non-essential
genes of virus are deleted and
exogenous genes are inserted

DISADVANTAGES OF VIRUSES AS
VECTOR IN GENE THERAPY
In all viral types, the vectors tend
not to disperse well in a targeted
tissue. Even when injected directly
into a tumor, they are prone to miss
some of the targeted cells.
In addition, their use does not allow
long-term gene expression.

NON-VIRAL VECTORS

PHYSICAL APPROACHES
Needle injection
Electroporation
Gene gun
Ultrasound
Hydrodynamic delivery
The simplest method of non-viral
transfection. Clinical trials carried out of
intramuscular injection of a naked DNA
plasmid have occurred with some
success; however, the expression has
been very low in comparison to other
methods of transfection.

LIPOSOMES
WHY NAKED DNA???
Lets wrap it in something safe
to increase transfection rate

Lipids are an obvious idea !

WHAT ARE LIPOSOMES?

They are artificial vesicles with a
phospholipid bilayer membrane.
They are self-closing spherical particles
where one or several lipid membranes
encapsulate part of the solvent in
which they freely float in their interior.
Liposomes are typically 5-10 m in
diameter with the phosopholipid
bilayer about 3 nm thick

Advantages of liposomes
Cheaper than viruses
No immune response

Especially good
for in-lung delivery (cystic fibrosis)

100-1000 times more plasmid DNA needed

for the same transfer efficiency as for viral vector

DNA delivery of genes by

liposomes
Fig. 19.20aa (TE Art)

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Liposome

Fig. 19.20ab (TE Art)

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Endosome

DNA carrying the

gene of interest

Target cell

By recombination, the DNA carrying

the gene of interest is integrated into
a chromosome of the target cell.

DNA-liposome complex is taken into

the target cell by endocytosis.
The liposome is degraded within the endosome
and the DNA is released into the cytosol.
The DNA is imported into the cell nucleus.

Integrated
gene
Nonviral approach

ARTIFICIAL CHROMOSOME
Another method is trying to
introduce a 47th chromosome.
It exists alongside the 46 others.
It could carry a lot of
information.
But how to get the big molecule
through membranes?

ETHICAL ISSUES
Who will have access to therapy?
Is it interfering with Gods plan?
Should people be allowed to use gene therapy to
enhance basic human traits such as height,
intelligence etc.?
Is it alright to use the therapy in the prenatal stage
of development in babies?

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