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Genetic Approaches forMalaria Control
Marcelo Jacobs-Lorena, PhD
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The Malaria Problem
At risk: >2,000,000,000 in 102 countries
Infected: >270,000,000
Clinically ill: >110,000,000 cases each year
Deaths: >2,000,000 each year• 90% in Africa• Mainly children 5-yr-old or younger
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From CDC web site
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Possible Weapons to Fight Malaria
1. Drugs that kill the parasite in humans
2. Insecticides that kill the mosquito vector
3. 3. Vaccines
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1. Drugs
Plasmodium falciparum is now largely resistant to chloroquine, which used to be the most effective drug.
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2. Insecticides: Mosquitoes Quickly Acquire Resistance
AfterBefore During
Biologic niche intact Mosquitoes return
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Mosquito Breeding
Photo: Marcelo Jacobs-Lorena
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Mosquito Breeding
Photo: Marcelo Jacobs-Lorena
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Mosquito Breeding
Photo: Marcelo Jacobs-Lorena
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3. Vaccine3. Vaccine……
… does not exist (yet).
It does not exist (yet)…
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The mosquito is an obligatory vectorThe mosquito is an obligatory vector(transmission by transfusion has no epidemiological significance)
Interference with parasite development Interference with parasite development will result in decreased transmissionwill result in decreased transmission
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Anopheles gambiae
CDC
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NEW APPROACH:NEW APPROACH:GENETIC MODIFICATION OF MOSQUITOESGENETIC MODIFICATION OF MOSQUITOES
1) GENETICALLY ENGINEER MOSQUITOESTO MAKE THEM RESISTANT TO PLASMODIUM
2) INTRODUCE THE REFRACTORY GENE(S)INTO MOSQUITO POPULATIONS
LESS TRANSMISSION LESS MALARIA
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Plasmodium development in the mosquito
Ghosh et al. (2003) Trends in Parasitology 19:94-101. Used with Permission.
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The use of a
PHAGE DISPLAY LIBRARY
to search for peptides with affinity to mosquito salivary gland and midgut epithelia
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Repeat selection 3 times
Enriched phage population
Dissect gut, open into a sheet, wash
Elute
Feeder
Midgut lumen
Elute
Repeat selection 3 times
Enriched phage population
Inject
Dissect, wash
Salivary gland
From Ghosh et al. Proc Nat Acad Sci USA 2001;98:13278-1328. Copyright 2001 National Academy of Sciences, USA. Used with permission.
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SM1 PEPTIDE
C C
Q
IA
FQ
SI
NP
R
BIOTIN
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SALIVARY GLANDS INCUBATED WITH BIOTINYLATED PEPTIDES
pSM1
FITC DAPI
unrelatedpeptide
From Ghosh et al. Proc Nat Acad Sci USA 2001;98:13278-1328. Copyright 2001 National Academy of Sciences, USA. Used with permission.
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THE SM1 PEPTIDE BINDS TO THE MIDGUT LUMENBUT NOT TO THE OUTSIDE
Open midgutsheet
FITC DIC optics
Closed (intact)midgut
From Ghosh et al. Proc Nat Acad Sci USA 2001;98:13278-1328. Copyright 2001 National Academy of Sciences, USA. Used with permission.
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SM1 Constructfor Mosquito Germ Line Transformation
Gut-specific & blood-induciblecarboxypeptidase promoter
Secretionsignal
[SM1]4
HA1epitope
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An. stephensi - piggyBac (3xP3-eGFP)eye-specific
promoter
WT
WT
TransgenicTransgenic
Transgenic
Larvae Adults
Images courtesy of Jacobs-Lorena
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Inhibition of oocyst formationin CP-[SM1]4 transgenic mosquitoes
Average of 9 experiments:
Oocysts/ Inhibitiongut
Non-trangenic 9382%
CP-[SM1]4 18(range: 69% - 95%)
Ito et al. (2002) Nature 417:452-455.
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Challenge: Driving Genes into Mosquito Populations
What drive mechanisms are being considered?
1. Transposable elements
2. Wolbachia
3. Meiotic drive
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An Alternative
Introduce genes into bacteriagenes into bacteria of the mosquito
midgut, instead of into the mosquito itself
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0
50
100
150
200
250
300
Mea
n oo
cyst
s/m
idgu
t
**
* p < 0.001
OmpA SM1-1 SM1-2
E. coliE. coliOmpAOmpA SM1
-- Feed Feed E.coliE.coli ( , or ) to ( , or ) to An. stephensiAn. stephensi mosquitoesmosquitoes-- After 24 h feed a After 24 h feed a P. bergheiP. berghei--infected blood mealinfected blood meal-- After 15 d count oocysts per gut (n > 100 mosquitoes) After 15 d count oocysts per gut (n > 100 mosquitoes)