Genetic Modified Organisms : Transgenic Mosquitoes

Presented by:Presented by: ANIL KUMARANIL KUMAR M.Sc. PHE iiM.Sc. PHE iind nd SemSem 2015-172015-17 VCRC(ICMR)VCRC(ICMR)

Your company slogan

Terminologies

TransgeneTransgene : : Gene or genetic material from one source added to other Gene or genetic material from one source added to other organism.organism.

Transgenesis :Transgenesis :the process of introducing an exogenous gene (transgene) the process of introducing an exogenous gene (transgene) into a living organism so that the organism will exhibit a new into a living organism so that the organism will exhibit a new property and transmit that property to its offspringproperty and transmit that property to its offspring..

Genetically modified Organisms :Genetically modified Organisms :Organisms (i.e. plants , animals or microorganisms) in which Organisms (i.e. plants , animals or microorganisms) in which the genetic material (DNA) has been altered in a way that the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural does not occur naturally by mating and/or natural recombination.recombination.

Your company slogan

Continued…

Transgenic Mosquitoes :Transgenic Mosquitoes :Mosquitoes in which genetic material (DNA) has Mosquitoes in which genetic material (DNA) has been altered in a way that does not occur naturally been altered in a way that does not occur naturally by mating and/or natural recombination.by mating and/or natural recombination.It is used in interruption of Vector Borne Disease It is used in interruption of Vector Borne Disease (VBD) cycle.(VBD) cycle.

Your company slogan

Need???

Inspite of more than 70 years of vector Inspite of more than 70 years of vector control operations disease burden is still control operations disease burden is still persisting.persisting.

Development of resistance against Chemicals Development of resistance against Chemicals used in vector control operations.used in vector control operations.

Environment and health hazards related to Environment and health hazards related to Chemical control of mosquito vectors.Chemical control of mosquito vectors.

Biological methods are not much effective.Biological methods are not much effective.

Your company slogan

Table of Contents

2. Methods2. Methods

3. Role in vector control 3. Role in vector control

4. Drawbacks4. Drawbacks

1. Introduction1. Introduction

Your company slogan

Introduction

For more than two decades, scientists have been For more than two decades, scientists have been working to develop genetically modified working to develop genetically modified mosquitoes for use as public health tools to mosquitoes for use as public health tools to prevent the transmission of mosquito borne prevent the transmission of mosquito borne diseases.diseases.

Recent advances in the development of genetically Recent advances in the development of genetically modified mosquitoes have raised hopes for the modified mosquitoes have raised hopes for the availability of new, potent and cost-effective tools availability of new, potent and cost-effective tools to aid in the fight against Dengue, Zika and to aid in the fight against Dengue, Zika and malaria.malaria.

Initial lab trails and small scale field trials have Initial lab trails and small scale field trials have shown a good result in shown a good result in Aedes Aedes aegyptiaegypti and and AnophelesAnopheles ablimanuablimanuss control.control.

FIRST RELEASE OF TRANSGENIC MOSQUITOES

Your company slogan

Strategies by transgenic mosquitoes

PopulationPopulationREPLACEMENTREPLACEMENTPopulation Population

suppressionsuppression

TransgenicTransgenic mosquitoesmosquitoes

Your company slogan

SELF –SELF – PERSISTINGPERSISTINGSelf - limitingSelf - limiting

Transgenicmosquitoe

s

Population

Population

suppression

suppression

Population

Population

REPLACEMENT

REPLACEMENT

Your company slogan

NATURAL ALLELE

SELF-LIMITING

PERS

ISTEN

SE

SELF-PERSISTAINING

INVA

SIVEN

ESS

Types of transgenesis in mosquitoesTypes of transgenesis in mosquitoes

Your company slogan

Sterile insect technique

The most strongly self-sustaining strategies The most strongly self-sustaining strategies invole the use of sterile insects.invole the use of sterile insects.

Mostly sterile-male strategies involve the use Mostly sterile-male strategies involve the use of heritable lethal genes or mutations.of heritable lethal genes or mutations.

Fully penetrant sterile/lethal traits the Fully penetrant sterile/lethal traits the modification is strongly self-limiting ,as it modification is strongly self-limiting ,as it disappears within a generation unless disappears within a generation unless additional modified insects are released.additional modified insects are released.

Irradiation, chemosterilization, transgenic Irradiation, chemosterilization, transgenic sterilization were the ancient methods of male sterilization were the ancient methods of male sterilization.sterilization.

Mosquitoes are modified by adding a gene that Mosquitoes are modified by adding a gene that produces a protein called tetracycline produces a protein called tetracycline repressible activator variant tTAV.repressible activator variant tTAV.

Your company slogan

Continued….

tTAV can able to act as a SWITCH that controls tTAV can able to act as a SWITCH that controls the activity of other genesthe activity of other genes

GM mosquito produces higher amount of this GM mosquito produces higher amount of this tTAV proteintTAV protein

As it activates its own gene, this will ties up As it activates its own gene, this will ties up some of the cell machinery.some of the cell machinery.

The modified gene is actually a lethal gene.The modified gene is actually a lethal gene. Tetracycline acts as an antidot to this gene.Tetracycline acts as an antidot to this gene. Hence the mosquitoes are supplimented with Hence the mosquitoes are supplimented with

tetracycline and mate with the wild mosquitoes.tetracycline and mate with the wild mosquitoes. The offsprings inherit the lethal gene and due The offsprings inherit the lethal gene and due

to the insufficiency of tetracycline in the field to the insufficiency of tetracycline in the field they will die.they will die.

Your company slogan

RIDL

Release of insects carrying a dominant lethal Release of insects carrying a dominant lethal genetic system (RIDL)genetic system (RIDL)

Two types :-Two types :- fsRIDL (female specific RIDL) bi-sex RIDL

It uses a lethal gene expressed in the zygot, It uses a lethal gene expressed in the zygot, rather than in the paternal parent.rather than in the paternal parent.

It releases transgenic mosquito homozygous It releases transgenic mosquito homozygous for the repressible dominant lethal gene.for the repressible dominant lethal gene.

In fsRIDL the lethal gene is female specific;In fsRIDL the lethal gene is female specific; Female offsprings will die Heterozygous male offsprigs will be alive If these heterozygous males mate half of their offspring

inherit the female lethal transgene

Your company slogan

Continued

In bi-sex RIDL the heterozygous In bi-sex RIDL the heterozygous offsprings are killed by the offsprings are killed by the inherited lethal gene.inherited lethal gene.

Your company slogan

Incompatible Insect Technique (IIT)

Wolbachia Wolbachia infected males were infected males were successfully used for vector control.successfully used for vector control.

Sterility can also be created by artificial Sterility can also be created by artificial infection with various strains of infection with various strains of Wolbachia.Wolbachia.

Wolbachia Wolbachia in effects conducts a sterile-in effects conducts a sterile-male attack in uninfected females.male attack in uninfected females.

For For Wolbachia, Wolbachia, all embryos produced by all embryos produced by an infected mother are infected.an infected mother are infected.

In addition, embryos are nonviable if In addition, embryos are nonviable if fertilized by a male carrying a fertilized by a male carrying a Wolbachia Wolbachia strain not carried by the femalestrain not carried by the female

Your company slogan

Homing Endonuclease Gene

HEGs are selfish DNA elements encoding proteins HEGs are selfish DNA elements encoding proteins (endonucleases) that recognise and cleave specific (endonucleases) that recognise and cleave specific DNA sequences of ˜20-30 nucleotides.DNA sequences of ˜20-30 nucleotides.

HEGs may drive recessive lethal or sterile alleles to HEGs may drive recessive lethal or sterile alleles to the population, potentially forcing it to extinction.the population, potentially forcing it to extinction.

Homing :- when the HEG is inserted into the Homing :- when the HEG is inserted into the potential site, repair of the HEG induced cut can potential site, repair of the HEG induced cut can copy the HEG-bearing allele.copy the HEG-bearing allele.

Occurrence of homing in the germ line causes Occurrence of homing in the germ line causes super-Mendelian inheritence, whereby more than super-Mendelian inheritence, whereby more than 50% of the offspring of a HEG heterozygote carry 50% of the offspring of a HEG heterozygote carry the HEG.the HEG.

It is of two types :-It is of two types :- X-shredder Sterile allele

Your company slogan

Continued…

If the HEGs cut X-linked sequence and the If the HEGs cut X-linked sequence and the HEG is expressed during HEG is expressed during spermatogenesis, this may results inspermatogenesis, this may results in Nonviable daughters The elimination of X-bearing system

If the X-shredder is Y-linked, all male If the X-shredder is Y-linked, all male offsprings carry it, which can lead to offsprings carry it, which can lead to invasion through meiotic driveinvasion through meiotic drive

Your company slogan

Transmission blocking vaccines

It consist of antibodies that are ingested It consist of antibodies that are ingested by the mosquito with the blood meal and by the mosquito with the blood meal and interfere with parasite development.interfere with parasite development.

Proteins expressed on the surface of Proteins expressed on the surface of gametes of parasites (eg., Pfs47/Pfs230) gametes of parasites (eg., Pfs47/Pfs230) and ookinetes (eg., Pfs25 & Pfs28) have and ookinetes (eg., Pfs25 & Pfs28) have been tested for such vaccines.been tested for such vaccines.

Antibodies against these protein bind to Antibodies against these protein bind to the parasite and block ookinete invasion the parasite and block ookinete invasion of the midgut epithelium.of the midgut epithelium.

Mosquito midgut antigens could be Mosquito midgut antigens could be targeted.targeted.

Your company slogan

Paratransgenesis

It is the genetic manipulation of commensal or symbiotic It is the genetic manipulation of commensal or symbiotic bacteria to alter the host’s ability to transmit a pathogen bacteria to alter the host’s ability to transmit a pathogen

It is an alternative means of preventing malaria It is an alternative means of preventing malaria transmissiontransmission

Bacteria can be engineered to express and secrete Bacteria can be engineered to express and secrete peptides or proteins that block parasite invasion or kill peptides or proteins that block parasite invasion or kill the parasite in the midgutthe parasite in the midgut

Gram-positive and gram negative bacteria including Gram-positive and gram negative bacteria including Escherichia, Alcaligenes, Pseudomonas, Serratia and Escherichia, Alcaligenes, Pseudomonas, Serratia and Bacillus Bacillus have been identified in the midgut of wild have been identified in the midgut of wild mosquito mosquito

Eg., Eg., An. stephensi An. stephensi fed with fed with E. ColiE. Coli that express a fusion that express a fusion protein of ricin and single chain antibody against protein of ricin and single chain antibody against P. P. Berghei Berghei ookinete surface protein and inhibited the oocyst ookinete surface protein and inhibited the oocyst formation upto 95%formation upto 95%

Your company slogan

Promoters and Effector genes

To produce GM culicine mosquitoes and Anopheline To produce GM culicine mosquitoes and Anopheline mosquitoes which interfere with mosquitoes which interfere with Plasmodium Plasmodium development promoters and effector genes can be useddevelopment promoters and effector genes can be used

Effecter genes products are those which hinder the Effecter genes products are those which hinder the parasite development.parasite development.

Promoters are those which drive the transgenesPromoters are those which drive the transgenes Single chain antibodies that recognize surface proteins Single chain antibodies that recognize surface proteins

show promise in interfering with parasite developmentshow promise in interfering with parasite development An anti-Pbs21 single chain antibody inhibited oocyst An anti-Pbs21 single chain antibody inhibited oocyst

formation by upto 95%formation by upto 95% A single chain antibody against A single chain antibody against P. gallinaceum P. gallinaceum

Circunsporozoite protein(CSP), expressed from a Sinbdis Circunsporozoite protein(CSP), expressed from a Sinbdis virus vector, reduced number of parasite in the salivary virus vector, reduced number of parasite in the salivary glands by 99%glands by 99%

Your company slogan

Continued…

Transgenic mosquitoes expressing bee venom Transgenic mosquitoes expressing bee venom PLA2 from the carboxypeptidase promoter PLA2 from the carboxypeptidase promoter reduced reduced P. berghei P. berghei oocyst formation by 87%.oocyst formation by 87%.

Transgenic Transgenic An. stephensi An. stephensi expressing PLA2 expressing PLA2 from AgAper1 promoter inhibited oocyst from AgAper1 promoter inhibited oocyst formation.formation.

An alternative strategy is to use synthetic An alternative strategy is to use synthetic molecules to interfere with parasite molecules to interfere with parasite development.development.

An. stephensi An. stephensi engineered with a synthetic engineered with a synthetic gene expressing a SM1 tetramer under the gene expressing a SM1 tetramer under the control of the carboxypeptidase promoter control of the carboxypeptidase promoter reduced the reduced the P. P. bbeghei eghei development.development.

Your company slogan

Refractory genes

These are genes that conferring the ability These are genes that conferring the ability to transmit the pathogen or parasiteto transmit the pathogen or parasite

Refractory genes may be used where Refractory genes may be used where modest prevalence and duration are modest prevalence and duration are adequateadequate

It can be used with a self-limiting driver It can be used with a self-limiting driver system eg., Killer-rescuesystem eg., Killer-rescue

Or without a driver system eg., Multi locus Or without a driver system eg., Multi locus assortment(MLA)assortment(MLA)

Dissociation of refractory gene:- loss of Dissociation of refractory gene:- loss of refractory gene DNA from a gene drive refractory gene DNA from a gene drive construct such that the refractory gene no construct such that the refractory gene no longer functionslonger functions

Your company slogan

Gene Drive Systems for Spreading Refractory Genes

Using Transposible elements (Tes)Using Transposible elements (Tes)One of the first gene drive systems to gain One of the first gene drive systems to gain widespread attentionwidespread attentionTEs replicate within a host genome and TEs replicate within a host genome and hence are inherited more frequently in hence are inherited more frequently in offspring’s genomeoffspring’s genomeThe increase in inheritance enables TEs to The increase in inheritance enables TEs to spread even in the presence of fitness cost to spread even in the presence of fitness cost to the hostthe hostThis has lead to widespread prevalence This has lead to widespread prevalence among many taxaamong many taxaVarious families of TEs represent 47% of the Various families of TEs represent 47% of the Aedes aegypti Aedes aegypti mosquito genomemosquito genome

Your company slogan

Medea elements

Maternal effect dominant embryonic arrest Maternal effect dominant embryonic arrest is a selfish gene composed of a toxin and is a selfish gene composed of a toxin and an antidote an antidote

A mother carrying Medea will express the A mother carrying Medea will express the toxin in her germline and kill her progenytoxin in her germline and kill her progeny

The offspring with medea allele produce The offspring with medea allele produce copies of the antidote that saving their copies of the antidote that saving their liveslives

In a heterozygous mother with one medea In a heterozygous mother with one medea allele and one non-medea allele,allele and one non-medea allele, half of the offsprings will inherit the medea allele and

survive Other half will inherit the non-medea and die

Your company slogan

Your company slogan

drawbacksdrawbacks

Self –limiting factor of transgenic mosquitoes will lead to periodic release of transgenic mosquitoes.

Periodic release of transgenic mosquitoes will be a costlier affair .

The SMT requires a very large number of males i.e, 5 sterile males to 1 wild female to mate.

Invasive genetic system requires more (wolbachia or UD) modified mosquitoes for their intial establishment.

About 3-4% of Sterile Modified Mosquito (SMM) carrying a dominant lethal gene escapes its activation. (Source: Department of Biosafety, Ministry of Natural Resources & Environment of Malaysia)

Use of antibiotics in GMM can lead to drug resistance.

Your company slogan

Selection of transgenic mosquitoes is a Selection of transgenic mosquitoes is a manual work.manual work.

Selection of transgenic male and female Selection of transgenic male and female larvae or pupae is manual work.larvae or pupae is manual work.

Mosquito fitness is a major concern.Mosquito fitness is a major concern.

Immigration into other areas.Immigration into other areas.

Community involvement, social and ethical Community involvement, social and ethical issues.issues.

Your company slogan

DARK SIDE OF

COIN

Your company slogan

OFF THE RECORDS…. (Coincidene Or .........!!)

OX513A was the name given to the modified male mosquitoes that were released in Brazil. They were bred in a lab in the UK and modified to require tetracycline, an antibiotic, to stay alive and destined to die if they didn’t get a regular supply of it.

Oxitech did two trials in Brazil i.e, 2012 and 2015.

Zika is not new to world, it was discovered in Uganda in 1947, microcephaly is a complication of zika which was first reported from the same area of Brazil where Oxitech did it trials.

Colombia, a neighbour of Brazil has recorded more than 20,000 cases of zika but has only reported a small increase of microcephaly related to zika. (up to April 16th 2016)

Epicentre of Zika in Brazil

Your company slogan

QUESTIONS YET TO BE ANSWERED...!!

Are we prepared for any epidemics because of gmm? Are we prepared against gmm, if it is used as bioterrorism

weapon? Ecological niche created by wiping a mosquito species will be filled

by other species so can gmm be used as only option? If we are wiping a mosquito species from an area it may lead to

infiltration on the mosquitoes from the neighboring areas, so release of gmm will it be a continuous process?

if you are releasing gmm (Aedes aegypti) in an area and in the same place any epidemic occurs because of other vector what control measures one will take ??

15 % of transgenic mosquitoes escapes the activation of lethal genes as tetracyclin (antidote) is also widely used in the field of agriculture , so if these mosquitoes will bite human then?? It may lead to resistance in mosquitoes too!!