aptamers targeting hiv-1 entry
DESCRIPTION
Aptamers Targeting HIV-1 Entry. Hazel Mufhandu PhD student Supervisor: Dr M Khati 2 nd Regional Synthetic Biology Forum 27 July 2012. Talk Outline. What are aptamers Aptamers against HIV-1 UCLA1 aptamer against HIV-1 subtype C. Proteins. Small Molecules. Whole organisms. - PowerPoint PPT PresentationTRANSCRIPT
Aptamers Targeting HIV-1 Entry
Hazel MufhanduPhD studentSupervisor: Dr M Khati2nd Regional Synthetic Biology Forum27 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
SmallMolecules
Proteins
Whole organismsCells
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?
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
Chemically produced and not prone to viral or bacterial contamination
Applications of Aptamers
What makes aptamers our tool of choice?
Limitations of aptamers
Small size makes them susceptible to renal filtration and thus shorter half-life
Unmodified aptamers are highly susceptible to serum degradation
Strategies to overcome limitations
Conjugation with PEG or cholesterol can increase circulating half-life
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’-flouro-pyrimidines
• UCLA1 RNA aptamer - Solid-phase synthesized
• Stabilized - inverted thymidine at 3’-end and dimethoxyltrityloxy-(CH2)6-SS-(CH2)6-phospho linker at 5’-end
Dey A K et al., RNA. 2005, 11:873Chen C et al., Virology. 2008, 381:46
B40 117nt
B40t 77nt
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
• TZM-bl cells
• Luminescent
reporter gene
assay
6 Primary Isolates
• PBMCs
• HIV-1 Gag p24
ELISA
5 Primary Isolates
• MDMs
• HIV-1 Gag p24
ELISA
HIV-1 pseudovirus neutralization in TZM-bl cells
ZM233
0.01 0.1 1 10 1000
25
50
75
100
Sensitive to UCLA1
% In
hibi
tion
CAP84
0.01 0.1 1 10 1000
25
50
75
Resistant to UCLA1
UCLA1 (nM)
Inhibited infection 29/35 (83%)
IC50 values <1 nMAverage 0.8 ± 0.9 nM• 27 = R5-tropic• 2 = X4-tropic • Subtype B X4-tropic
• IC50 = 0.17 nM
UCLA1 neutralize HIV-1 subtype C pseudoviruses
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-tropic1: X4 tropic
Neutralization in PBMCs
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
TM3IC80 = 63nM
3.6 11 33 100 Control0.0
2.5
5.0
7.5
10.0
UCLA1 (nM)
p24
(ng/
ml)
Du422IC80 = 81 nM
3.6 11 33 100 Control0.0
2.5
5.0
7.5UCLA1Virus Control
UCLA1 (nM)
p24
(ng/
ml)
*** p < 0.005 ** p < 0.01
RP1IC80 = 82 nM
3.7 11 33 100 Control0
10
20
30
40
UCLA1 (nM)
p24
(ng/
ml)
CAP63IC80 = 94 nM
3.6 11 33 100 Control0
5
10
15
UCLA1 (nM)
p24
(ng/
ml)
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
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Du422IC80 = 6 nM
3.6 11 33 100
Contro
l0.0
2.5
5.0
7.5
UCLA1 (nM)
P24
(ngm
l)COT9
IC80 = 29 nM
3.6 11 33 100
Contro
l0
1
2UCLA1 (nM)Virus Control
UCLA1 (nM)
p24
(ng/
ml)
*** p < 0.001 * p < 0.05NS: not significant
SW14IC80 = 26 nM
3.6 11 33 100
Contro
l0
1
2
3
UCLA1 (nM)
p24
(ng/
ml)
ADAIC80 = 30 nM
3.6 11 33 100
Contro
l0
10
20
30
40
UCLA1 (nM)
p24
(ng/
ml)
NS
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
•KD = 5 fold ↑Core gp120
•KD = 3 fold ↑
∆V1/V2
•KD = 3 fold↑
∆V3
Mapping UCLA1 binding sites on gp120
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
•KD = 3 fold ↑
I420R
•KD = 3 fold ↑
D368R
Mapping UCLA1 binding sites on gp120
0
10
20
30
40
50
60 IC50 > 10 nMIC50 < 10 nM
IC50
(nM
)
ConC env pseudovirus single point mutations
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
UCLA1 binding epitopes on gp120
K305I307
R308
H330
L125
L369
R419
K121
K305I307
CD4bs
CoRbs
V3 loop
90°
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Combination therapy
• UCLA1 + T20
• UCLA1 + IgG1b12Combinations
• 0.3 – 0.7 = Synergy
• 0.7 – 0.85 = Moderate synergy
• 0.9 – 1.1 = Additive
• >1.1 = Antagonistic
Combination Index = (D)1/(Dx)1
(D)2/(Dx)2
Combination therapy
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
• 4 synergism CI range 0.5 – 0.7
• 1 additiveCI = 0.93
• 1 Antagonism CI = 1.62
Tested 6 Env-pseudoviruses
UCLA1 + b12
Combination therapy
Du156 T20
0.01 0.1 1 10 1000
25
50
75
100
ZM249 T20
0.01 0.1 1 10 1000
25
50
75
100UCLA1+T20UCLA1T20
Du172 b12
0.01 0.1 1 10 1000
25
50
75
100
% In
hibi
tion
SW7 b12
0.01 0.1 1 10 1000
25
50
75
100
M (Log)
UCLA1+b12UCLA1b12
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
UCLA1 synergism
•11 fold less T20
•5 fold less b12
•5 fold less UCLA1
Dose Reduction Index
= A A+B
H T Mufhandu et al., J. Virol. May 2012, 86(9):4989
Escape Mutations
Tested 2 primary isolates in PBMC in ↑ [UCLA1]
No Escape mutations: Du422
(R5 tropic)RP1 virus propagated
12 wksIC70 ↑ 7x: 46 – 308 nM
Du422 virus
propagated 9 wksIC70 ↑ 4x: 33 – 119 nM
6 Escape mutations:RP1
(X4-tropic)
Initial [UCLA1] @ IC70
p24 assay7 day intervals gp120 sequence
analysis: test and control
cultures
UCLA1 Escape Mutations
R202T
F223Y
R322Q
P369L
N410S
K476R
Adapted from structure 2B4C (Huang et al., 2005)
Concluding remarks: UCLA RNA aptamer
HIV-1 Entry inhibitor
Broad spectrum potency against
subtype C isolates
Non-cytotoxic
High binding affinity
• HIV-1 ConC gp120
Mapped binding sites
CoRbs (V3 loop base)
Bridging sheet (V1/V2 loop)
CD4bs (C3, C4, C5 regions)
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 Cohort Viral Tropism
26x Acutely infected Adults CAPRISA 002 Acute Infection study
R5Subtype C Reference Panel –Durban, Zambia, Caprisa
isolates
2x Chronically infected Adults TB clinic
X4HxB2 – Subtype B
7x Chronically infected Paediatrics Chris Hani Baragwanath hospital
6 - R5
1 - X4
Subtype C consensus sequence clone (ConC) 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
Thromotic microangiopathies and carotid artery disease
Phase II
NU172 (ARCA biopharma)
Thrombin Cardiopulmonary bypass to maintain steady state of anticoagulation
Phase II
ARC1905 (Ophthotech)
Complement component 5
Age-related macular degeneration
Phase I
E10030 (Ophthotech)
Platelet-derived growth factor
Age-related macular degeneration
Phase I
NOX-A12 (NOXXON Pharma)
CXCL12 Multiple myeloma and non-Hodgkin’s lymphoma
Phase I
NOX-E36 (NOXXON Pharma)
CCL2 Type 2 diabetes, diabetic nephropathy
Phase I
Reviewed by Keefe A.D, et al., Nature Reviews. July 2010, 9:537.