Genetic manipulation of vector competence

1
Genetic manipulation of vector competence
Abraham Eappen and Marcelo Jacobs-Lorena
Johns Hopkins University Bloomberg School of
Public Health Dept. of Molecular Microbiology
Immunology Malaria Research Institute Baltimore,
Maryland 21205 U.S.A.
2
RATIONALE
3
Malaria kills gt1 million persons/year and...
the number of deaths is increasing!
4
What weapons are available to fight malaria?
5
THE ARSENAL IS VERY SMALL
6
RATIONALE
The mosquito is an obligatory vector
Interference with parasite development will
result in decreased transmission
7
GENETIC MODIFICATION OF MOSQUITOES
1) GENETICALLY ENGINEER MOSQUITOES TO MAKE THEM
RESISTANT TO PATHOGEN
8
HOW?
9
Plasmodium development in the mosquito
Ghosh et al. (2003) Trends in Parasitology
1994-101.
10
The oocyst bottleneck
Number of parasites
Gametoc.
Oocyst
Sporoz.
11
Genetic modification of mosquitoes WHAT IS
NEEDED?
Stable germ line transformation
Tissue-specific promoters
Effector genes that interfere with Plasmodium
development in the mosquito
12
TRANSFORMATION
13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
PROMOTERS
17
Carboxypeptidase promoter
18
Peritrophic matrix protein 1 (PM1) expression
19
Vitellogenin promoter
20
Strong salivary gland promoter tested in
transgenic mosquitoes (coming soon) S. Yoshida,
Jichi Medical School,Japan
Salivary gland promoter
21
EFFECTORS
1) Effectors administered exogenously to
mosquitoes
22
(No Transcript)
23
(No Transcript)
24
The use of a
PHAGE DISPLAY LIBRARY
to search for peptides with affinity to mosquito
salivary gland and midgut epithelia
25
(No Transcript)
26
(No Transcript)
27
SM1 PEPTIDE
I
F
A
Q
R
S
Q
I
P
C
C
N
BIOTIN
Ghosh et al. (2001) Proc. Nat. Acad. Sci. USA
9813278-13281.
28
SALIVARY GLANDS INCUBATED WITH BIOTINYLATED
PEPTIDES
pSM1
FITC
DAPI
unrelated peptide
Ghosh et al. (2001) Proc. Nat. Acad. Sci. USA
9813278-13281..
29
THE SM1 PEPTIDE BINDS TO THE MIDGUT LUMEN BUT NOT
TO THE OUTSIDE
Open midgut sheet
DIC optics
FITC
Closed (intact) midgut
Ghosh et al. (2001) Proc. Nat. Acad. Sci. USA
9813278-13281..
30
EFFECTORS
2) Effectors expressed in mosquitoes
31
(No Transcript)
32
SM1 construct for mosquito germ line
transformation
Gut-specific blood-inducible carboxypeptidase
promoter
SM14
HA1 epitope
Secretion signal
33
An. stephensi - piggyBac (3xP3-eGFP)
eye-specific promoter
Transgenic
WT
Transgenic
Transgenic
WT
Adults
Larvae
34
(SM1 peptide)
35
(No Transcript)
36
Virus titers of individual mosquitoes fed an
infected blood meal HWE control Carb77
transgenic
37
HOW FIT?
38
For fitness measurements, outcrossing is
important
Gene with no fitness load
Recessive mutation with fitness load WHEN
HOMOZIGOUS
39
Cage experiments MOSQUITOES FED ON NON-INFECTED
MICE
Expt. 1
Expt. 2


wild type
wild type
transgenic
transgenic
(generation)
(generation)
40
Marrelli et al.,Proc Natl Acad Sci U S A. (2007)
1045580-3.
Cage experiments MOSQUITOES FED ON INFECTED MICE
41
CHALLENGE DRIVING GENES INTO MOSQUITO POPULATIONS
42
What drive mechanisms are being considered?
Transposable elements
Wolbachia
Meiotic drive
43
POINTS TO CONSIDER
44
TRANSGENESIS
Need to improve efficiency of An. gambiae
transformation. Establishment of repository
centers for transgenic lines is desirable.
45
PROMOTERS
Promoters that are useful for most applications
are available.
46
EFFECTORS
Best targets midgut stages or late ookinete
because of low numbers, salivary gland (?)
because of length of exposure. For viruses,
midgut is ideal. No ideal effector has yet
been found SM1 not functional for P.
falciparum, PLA2 has fitness load, resistant
variants may emerge with monoclonals.
Antibacterial peptides, chitinase inhibitors, or
enhancement of native mosquito defense systems,
are possible additional effectors. Cargo
size. Multiple effectors MUST be used for
long-term effectiveness. However, the larger the
construct, the more difficult it is to drive it
intact through large number of generations. For
instance, 2 effectors 1 transformation marker
1 engine (e.g., transposase) 4 genes _at_ 3
kb/gene 12 kb.
47
DRIVING GENES INTO WILD POPULATIONS
TRANSPOSABLE ELEMENTS? WOLBACHIA? MEIOTIC
DRIVE? PARATRANSGENESIS?
48
Mission accomplished
Future
Time
Present
Past
Lab
Field
49
Acknowledgements
Dr. Jacobs-Lorena
50
Thank you for attending this presentation