Aptamers Targeting HIV-1 Entry

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


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


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


UCLA1- gp120 binding Kinetics

UCLA1- gp120 binding Kinetics


Mapping UCLA1 binding sites on gp120


•KD = 5 fold ↑Core gp120

•KD = 3 fold ↑

∆V1/V2

•KD = 3 fold↑

∆V3



•KD = 3 fold ↑

I420R

•KD = 3 fold ↑

D368R


0

10

20

30

40

50

60 IC50 > 10 nMIC50 < 10 nM

IC50

(nM

)

ConC env pseudovirus single point mutations


UCLA1 binding epitopes on gp120

K305I307

R308

H330

L125

L369

R419

K121

K305I307

CD4bs

CoRbs

V3 loop

90°


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


• 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


UCLA1 synergism

•11 fold less T20

•5 fold less b12

•5 fold less UCLA1

Dose Reduction Index

= A A+B


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


Phase I

E10030 (Ophthotech)

Platelet-derived growth factor


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.

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Aptamers Targeting HIV-1 Entry