Transcript Slide 1

Aptamers Targeting HIV-1 Entry
Hazel Mufhandu
PhD student
Supervisor: Dr M Khati
2nd Regional Synthetic Biology Forum
27 July 2012
Talk Outline
 What are aptamers
 Aptamers against HIV-1
 UCLA1 aptamer against HIV-1 subtype C
What is an aptamer?
An aptamer is an artificial nucleic acid ligand that is evolutionary engineered
in vitro using the SELEX (Systematic Evolution of Ligands by Exponential
Enrichment) process for specific binding to a variety of targets
Proteins
Small
Molecules
Cells
Whole organisms
How do we make aptamers?
SELEX Process
The resulting enriched
oligonucleotide ligands are
referred to as APTAMERS
derived from the Latin word
aptus meaning “to fit”
What makes aptamers our tool of choice?
Chemically produced and not prone to
viral or bacterial contamination
Have molecular recognition properties of
antibodies
Smaller size (8-15 kDa) allow efficient entry
into biological compartments
Recognize targets with high affinity and
specificity
Non-immunogenic & non-toxic in therapeutic
applications
Conjugation chemistries for attachment of
dyes, reporter molecules or functional groups
can be easily introduced during synthesis
Applications of Aptamers
What makes aptamers our tool of choice?
Limitations of aptamers
Strategies to overcome
limitations
 Small size makes them susceptible to
renal filtration and thus shorter half-life
 Conjugation with PEG or cholesterol
can increase circulating half-life
 Unmodified aptamers are highly
susceptible to serum degradation
 Chemical modifications incorporated
into the sugars or internucleotide
phosphodiester linkages enhance
nuclease resistance
Aptamers against HIV-1 entry
Aptamers against HIV-1 entry
• B40 stabilized with 2’-flouropyrimidines
B40t 77nt
B40 117nt
Dey A K et al., RNA. 2005, 11:873
Chen C et al., Virology. 2008, 381:46
•UCLA1 RNA aptamer - Solid-phase
synthesized
• Stabilized - inverted thymidine at
3’-end and dimethoxyltrityloxy(CH2)6-SS-(CH2)6-phospho linker
at 5’-end
Rationale of current UCLA1 study
To test the efficacy of
UCLA1 aptamer
as an entry inhibitor of
HIV-1 subtype C infection
Neutralization Methods
35 Env
Pseudoviruses
6 Primary
Isolates
5 Primary
Isolates
• TZM-bl cells
• PBMCs
• MDMs
• Luminescent
• HIV-1 Gag p24
• HIV-1 Gag p24
reporter gene
assay
ELISA
ELISA
HIV-1 pseudovirus neutralization in TZM-bl cells
Resistant to UCLA1
Sensitive to UCLA1
CAP84
ZM233
75
% Inhibition
100
75
50
50
25
25
0
0.01
0.1
1
10
100
0
0.01
UCLA1 (nM)
0.1
1
10
100
UCLA1 neutralize HIV-1 subtype C pseudoviruses
Inhibited infection 29/35 (83%)
IC50 values <1 nM
Average 0.8 ± 0.9 nM
• 27 = R5-tropic
• 2 = X4-tropic
• Subtype B X4-tropic
• IC50 = 0.17 nM
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
UCLA1 neutralize primary isolates in PBMC
Tested 6 primary isolates in PBMC
HIV-1 p24 antigen assay
Inhibited 4/6 isolates
IC80 average = 80 nM
3: R5-tropic
1: X4 tropic
Neutralization in PBMCs
Du422
IC80 = 81 nM
TM3
IC80 = 63nM
7.5
UCLA1
Virus Control
7.5
5.0
p24 (ng/ml)
p24 (ng/ml)
10.0


2.5
5.0
*** p < 0.005
** p < 0.01

2.5



0.0
0.0
3.6
11
33
100
3.6
Control
33
100
UCLA1 (nM)
UCLA1 (nM)
RP1
IC80 = 82 nM
CAP63
IC80 = 94 nM
40
Control
15
30
p24 (ng/ml)
p24 (ng/ml)
11
20

10
10
5


0
0
3.7
11
33
100
UCLA1 (nM)
Control
3.6
11
33
100
UCLA1 (nM)
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Control
UCLA1 neutralize primary isolates in MDMacrophages
Tested 5 primary isolates in MDM
incl. 1 subtype B virus
HIV-1 p24 antigen assay
Inhibited 3/4 subtype C isolates
IC80 average = 20 nM
1 subtype B - IC80 = 30nM
All R5-tropic
Neutralization in MDMs
COT9
IC80 = 29 nM
Du422
IC80 = 6 nM
2
p24 (ng/ml)
P24 (ngml)
7.5
5.0
NS
2.5
UCLA1 (nM)
Virus Control
*** p < 0.001
* p < 0.05
1
NS: not significant

0.0
l
C
UCLA1 (nM)
UCLA1 (nM)
SW14
IC80 = 26 nM
ADA
IC80 = 30 nM
3
p24 (ng/ml)
40
2
1


30
20

10


0

UCLA1 (nM)
UCLA1 (nM)
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
C
on
tro
l
10
0
33
11
3.
6
ro
l
on
t
C
10
0
33
11
6
0
3.
p24 (ng/ml)
on
tro
10
0
33
11
3.
6
ro
l
on
t
C
10
0
33
11
3.
6
0
Cytotoxicity Assays
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
UCLA1- gp120 binding Kinetics
UCLA1- gp120 binding Kinetics
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Mapping UCLA1 binding sites on gp120
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Core
gp120
• KD = 5
fold ↑
∆V1/V2
• KD = 3
fold ↑
∆V3
• KD = 3
fold↑
Mapping UCLA1 binding sites on gp120
I420R
• KD = 3 fold ↑
D368R
• KD = 3 fold ↑
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Mapping UCLA1 binding sites on gp120
ConC env pseudovirus single point mutations
IC50 > 10 nM
IC50 < 10 nM
60
IC50 (nM)
50
40
30
20
10
0
C
C
on
t
w
I
P
A
1A 25A 05A 07A 08A 30Y 32A
1A 23A 30A 40E 68V 71E 74A 76A
65 69
5M 19
2
2
3
7
3
3
3
4
4
4
4
I3
S
I4
L1
L3
F4
K1
K3
K4
V4
A4
R
H
N
R
D
R
G
S3
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
UCLA1 binding epitopes on gp120
CD4bs
CoRbs
L369
H330
R419
K121
L125
K305
I307
V3
loop
K305
I307
R308
90°
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Combination therapy
• UCLA1 + T20
Combinations
• UCLA1 + IgG1b12
• 0.3 – 0.7 = Synergy
Combination Index =
(D)1/(Dx)1
(D)2/(Dx)2
• 0.7 – 0.85 = Moderate synergy
• 0.9 – 1.1 = Additive
•
>1.1
= Antagonistic
Combination therapy
Tested 6
Env-pseudoviruses
UCLA1 + T20
Tested 6
Env-pseudoviruses
UCLA1 + b12
• 5 synergism
CI range 0.13 – 0.46
• 1 moderate synergism
CI = 0.84
• 4 synergism
 CI range 0.5 – 0.7
• 1 additive
CI = 0.93
• 1 Antagonism
 CI = 1.62
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Combination therapy
ZM249 T20
Du156 T20
100
100
75
75
50
50
25
25
0
0.01
0.1
1
10
100
0
0.01
UCLA1+T20
UCLA1
T20
0.1
Du172 b12
% Inhibition
10
100
SW7 b12
100
100
75
75
50
50
25
25
0
0.01
1
0.1
1
10
100
0
0.01
UCLA1+b12
UCLA1
b12
0.1
1
M (Log)
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
10
100
UCLA1 synergism
Dose Reduction
Index
= A
A+B
•11 fold less T20
•5 fold less b12
•5 fold less UCLA1
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Escape Mutations
Tested 2 primary isolates in PBMC in ↑ [UCLA1]
Initial [UCLA1] @ IC70
p24 assay
7 day intervals
RP1 virus propagated
12 wks
IC70 ↑ 7x: 46 – 308 nM
Du422 virus
propagated 9 wks
IC70 ↑ 4x: 33 – 119 nM
gp120 sequence
analysis:
test and control
cultures
No Escape mutations:
Du422
(R5 tropic)
6 Escape mutations:
RP1
(X4-tropic)
UCLA1 Escape Mutations
R322Q
N410S
P369L
R202T
K476R
F223Y
Adapted from structure 2B4C (Huang et al., 2005)
Concluding remarks: UCLA RNA aptamer
 HIV-1 Entry inhibitor
 Non-cytotoxic
 Broad spectrum potency against
 High binding affinity
subtype C isolates
Mapped binding sites
 CoRbs (V3 loop base)
 Bridging sheet (V1/V2 loop)
 CD4bs (C3, C4, C5 regions)
•
HIV-1 ConC gp120
 Synergy: HIV-1 entry inhibitors
 Escape mutations: > 5 fold IC70 of
UCLA1
 Further testing of UCLA1 in clinical studies
 As potential new HIV-1 entry inhibitor drug
 Against subtype C viruses
Aptamer Group
Acknowledgements
Project Supervision
• Dr Makobetsa Khati (CSIR
Biosciences)
• Prof Lynn Morris (NICD)
NICD HIV/AIDS Lab
• Elin Gray
• Maphuti Madiga
• Nancy Tumba
• Alex Kabamba
• Mary Phoswa
Collaborations
• Lynn Morris (NICD)
• William James (Oxford University)
Reagents
• Ian McGowan (NIH)
• William James (Oxford University)
• Brian Sproat (ATDBio, University of
Southampton)
FRC Individual Grant
Thank you
Can UCLA1 neutralize HIV-1 pseudoviruses?
Patients
26x Acutely infected Adults
Cohort
CAPRISA 002 Acute Infection
study
Subtype C Reference Panel –
Durban, Zambia, Caprisa
isolates
2x Chronically infected Adults
Subtype C consensus sequence clone
(ConC)
R5
TB clinic
HxB2 – Subtype B
7x Chronically infected Paediatrics
Viral Tropism
Chris Hani Baragwanath
hospital
X4
6 - R5
1 - X4
R5
Reviewed by Zhou and Rossi, Oligonucleotides. 2011, 21(1).
Larry Gold et al., Plos One. 2010, 5(12).
Name (Company)
Target
Indication
Current Phase
Pegaptanip
sodium/Macugen
(Pfizer/Eyetech)
Vascular Endothelial
growth factor
Age-related macular
degeneration
Approved in the US
and EU
AS1411/AGRO001
(Antisoma)
Nucleolin
Acute myeloid
leukemia
Phase II
REG1/RB006
plus RB007
(Regado
Biosciences)
Coagulation factor
IXa
Percutaneous
coronary
intervention
Phase II
ARC1779
(Archemix)
A1 domain of von
Willebrand factor
Phase II
NU172
(ARCA biopharma)
Thrombin
ARC1905
(Ophthotech)
Complement
component 5
Thromotic
microangiopathies
and carotid artery
disease
Cardiopulmonary
bypass to maintain
steady state of
anticoagulation
Age-related macular
degeneration
E10030
(Ophthotech)
Platelet-derived
growth factor
Age-related macular
degeneration
Phase I
NOX-A12
(NOXXON Pharma)
CXCL12
Phase I
NOX-E36
(NOXXON Pharma)
CCL2
Multiple myeloma
and non-Hodgkin’s
lymphoma
Type 2 diabetes,
diabetic
nephropathy
Phase II
Phase I
Phase I
Reviewed by Keefe A.D, et al., Nature Reviews. July 2010, 9:537.