LETHAL GENES ANDGENE THERAPY

BY ANILA AND POOJA

LETHAL GENESGenes 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 Cuénot 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 ALLELESLethal 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 final stage of life are known as late onset allele.

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 alleles kills organisms in homozygous condition only.

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.

• This occurs because homozygous creeper chickens die.

HUNTINGTON’S 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 the HTT gene cause Huntington disease. The HTT gene provides instructions for making a protein called huntingtin.

• HH Individual dies of Huntington’s disease

• Hh Individual dies of Huntington’s 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 development of embryo cannot be continued - the embryo dies.

Let's say that an allele a is recessive and codes for a completely dysfunctional form of a protein essential for bone growth, and A is 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 CF lung

Dilated crypts filled with mucus and bacteria.

Normal alveolar appearance

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 AY ), it kills the individual in early embryonic state.

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 born due to lethal effect of AY gene.

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.

• Regulation of particular gene could be altered(degree to which a gene could be turned on or off).

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 next generations.

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

1. Identification of the defective gene.2. Cloning of normal healthy gene.3. Identification of target cell / tissue / organ.4. Insertion of the normal functional gene into the

host DNA5. Transferred gene (TRANSGENE) encodes &

produces proteins6. The Proteins encoded by Transgene corrects the

disorder

APPROACHES TO GENE THERAPY

EX-VIVO 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 re-introduced 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:In vivo approach:

(Direct Gene Transfer)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

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 in retroviruses is in the form

of RNA molecules.• They can transfect dividing cells because they can pass

through the nuclear pores of mitotic cells.• Retroviruses are useful for ex vivo delivery 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 integrates into

the human genome using integrase

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

expression

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 APPROACHESNeedle injectionElectroporationGene gunUltrasoundHydrodynamic 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???

Lipids – are an obvious idea !

Lets’ wrap it in something safe to increase transfection rate

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 µm 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

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

LiposomeDNA carrying thegene of interest

Target cell

The liposome is degraded within the endosomeand the DNA is released into the cytosol.

DNA-liposome complex is taken intothe target cell by endocytosis.

The DNA is imported into the cell nucleus.

Fig. 19.20aa (TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Nonviral approach

Endosome

Integratedgene

By recombination, the DNA carryingthe gene of interest is integrated intoa chromosome of the target cell.

Fig. 19.20ab (TE Art)

DNA delivery of genes by liposomes

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 God’s 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?

gene

Alright, tell me how

you want to change!