Structural biology for the optimization of gene …oge.med.ufl.edu/courses/gms 6001/IDP_MAM_9_18_2012.pdf · Structural biology for the optimization ... Structural biology for the

1

Structural biology for the optimization

of gene therapy vectors

COM IDP Ground Rounds 9/18/12

Mavis Agbandje-McKenna

Biochemistry & Molecular Biology

LG-181 (office), LG-171 (lab)

[email protected]

http://www.mbi.ufl.edu/facilities/msg

Structural biology for the optimization

of gene therapy vectors

COM IDP Ground Rounds 9/18/12

Presentation Outline

• Viruses

• The AAVs

• Gene Therapy, Challenges

• The AAV Capsid Structure – 3D

• Examples of Structure-based vector engineering

Viruses

• Genome is infectious the infectious material

• Protein/lipid shell assembles around the genome

• Shell dictates cell/tissue tropism

• Tropism also dictated by cell surface molecules

• Glycans (carbs) are major components of cell surface molecules – protein/lipid

• Shell dictates antigenic reactivity – control (vaccines)

• Some controlled by small molecule inhibitors

Geminiviridae

Viruses

Microviridae

?

2

Viruses

• Genome is infectious the infectious material

• Protein/lipid shell assembles around the genome

• Shell dictates cell/tissue tropism

• Tropism also dictated by cell surface molecules

• Glycans (carbs) are major components of cell surface molecules – protein/lipid

• Shell dictates antigenic reactivity – control (vaccines)

• Some controlled by small molecule inhibitors

Geminiviridae

Viruses

Microviridae

DNA OR RNA

Protein (and Lipid)

Gene Therapy• “A technique for correcting

defective genes responsible for disease development“

• Vehicles for gene delivery

• Lipososmes, naked DNA

• Viral vectors

–Adenoviruses

–Retroviruses/Lentiviruses

–Adeno-associated viruses

AAV GT

Cytoplasm

Replication Transcription

RibosomeTherapeutic

Protein

Nucleus

PM

AAV Serotypes and Gene Delivery

Adapted From Gao et al., JV 2004

Clade E AAV8

Clade D AAV7

Clade F AAV9

Clade C

AAV2-3

Clade A

AAV1-6

Clade B AAV2

AAV4AAV5

Non-pathogenic and non-toxic

Over 100 genotypes isolated

Grouped into antigenically

distinct clades & clones

~57-99% identical at aa level

Can package foreign genes

Representative members under

development for gene delivery

Pseudo-typed vectors show

capsid-associated

differential tissue tropism &

transduction efficiency

Capsid - antigenic reactivity

AAV2 vs. AAVs

AAV Basics

3











capsid-associated




Clade A: AAV1 / AAV6

Clade B: AAV2

Clade C: AAV2 –AAV3 hybrid

Clade D: AAV7

Clade E: AAV8

Clade F: AAV9

Clonal

Isolates:

AAV4

AAV5

Similarity

AAV1 83.70%

AAV3 89.16%

AAV4 57.30%

AAV5 57.06%

AAV6 83.65%

AAV7 82.39%

AAV8 82.20%

AAV9 81.21%

AAV2 vs. AAVs

AAV Basics











capsid-associated




AAV Basics

1

4

5

2

Lung

Brain

Retina

Retina

Brain

Broad

Muscle

Muscular

Dystrophy

Emphysema

Cystic Fibrosis

CNS diseases

Blindness

Emphysema

Diabetes

Blindness

Blindness 8

Muscle

Liver

Muscle

Liver

Muscle

Muscular

Dystrophy

Hemophilia

Diabetes

Hemophilia

Heart Disease

Liver

Hemophilia

Diabetes

Heart Failure

6

7

9











capsid-associated




AAV Basics


Transduction Phenotypes

Tran

sgen

e

2

2

987

5

2

2

1

9

9

9

8

8

8

7

7

7

5

5

1

1

1

4

Gene Delivery/ImprovementVirus+ reporter gene

+

REQUIRES

UNDERSTANDING

OF THE BASIC BIOLOGY

OF THE VECTOR

AAV GT

Virus

+

Non-human Primates

4.7 kb linear ssDNA genome is

packaged as plus and minus

strands in separate virus

particles – rep and cap

genes

Termini consist of short inverted

terminal repeats (ITRs),

which function as the

packaging signal for AAV.

Three different promoters: P5,

P19 and P40.

(From Blechacz and Russell, Expert Reviews in Molecular

Medicine: Vol. 6; Issue 16.)

AAV Viral Genes and Gene Products

Capsid contains 60 copies (in total) of VP1, VP2, and VP3, ratio 1:1:10, ~260Å

VP1 – N terminal unique region – PLA2 activity required for infectivity

&Nuclear localization

Common VP region has multiple functions:

Receptor/Co-receptor recognition/Endosomal trafficking;

Nuclear entry/egress;

Capsid assembly/DNA packaging;

Antibody recognition/neutralization

Rep Proteins facilitate genomic integration, replication, and packaging

AAV Basics

AAV Life Cycle

Latent LyticAAV

Adenovirus

(Or Herpes Virus)

Adenovirus

(Or Herpes Virus)

Chr 19

integration

AAV Basics

5

Recombinant AAV vectors

Transgene of InterestTR TR

Plasmid

cell

AAV

AD

Transgene

AAV GT

E.g. AAVs in the Clinic

Adapted from Mingozzi F and High K, 2011 NATURE REVIEWS | GENETICSBowles et al., 2012 Mol. Ther

AAV GT

GENE THERAPY CHALLENGES• Issues surrounding AAV viral vectors in gene therapy

– specific tissue targeting, transient or low expression

– immunogenicity• Directed against AAV capsid and therapeutic gene

– Originally thought to have low immunogenicity – mouse studies

– Current studies show pre-existing B-cell antibody against the capsid and T-cell responses against capsid peptides in large animal models and human trials

– Human population - 50-95% seropositive, 18-60% Nabs

– Low titers of pre-existing NAbs prevents re-administration

• Need to understand tissue tropism and transduction determinants of AAVs– Enable engineering of targeted tissue tropism and improved


• Need to understand antigenic structure of AAV capsids– Enable engineering of second generation vectors

• Have similar tropism to parental viruses

• Can evade pre-existing immune response

AAV GT

6

Schematic of AAV Lytic Infection (Very Simplified)

entry1

2 internalization

3

Early endosome

pH 6

4

Late endosome

pH 5 5

Lysosome

pH 4

Recycling

endosome

Golgi

6

uncoating and

genome release

genome

replication

7

Nucleus

Cytoplasm

Extracellular space

8

genome

transcription

9

protein

expression

0/10capsid

assembly

11

genome

packaging

12egress

Endoplasmic

Reticulum

Legend (NOT to scale)

Parvovirus

Glycan

Glycoprotein

Microtubules

Clathrin

Dynamin

Actin

Ribosome

Proteasome

Nuclear pore complex

Viral genome

vRNA transcripts

Structural proteins (VP)

Viral intermediates

Nonstructural proteins (NS)

Oligomerized NS

Replication intermediate

Antibody

13

antibody

recognition

(Halder et al, 2012)

AAV2 & AAV5

Levels of

Protein Structure

I-Primary II-Secondary

III-Tertiary

Van der Waal

H-bonding

Disulphides

Structure = Function

IV-Quaternary

AAV Serotype Transduction and Serotype



Tra

nsg

en

e

2

2

9875

2

2

1

9

9

9

8

8

8

7

7

7

5

5

1

1

1

• Build a 3D Library of Representative

Members: AAV1-AAV9

• Functionally Annotate the VP/Capsid

w.r.t.

• Receptor attachment determinants

• Transduction determinants

•Antigenic determinants

• Use information for engineering second

generation vectors with improved

efficacy for clinical use

•E.g. By capsid modifications

GOALS:

AAV Basics

7

Cryo-EM and Image Reconstruction 101 Methods

1 2 3

4 5 10

< 10Å

F386

L388C387

F385

E389

X-ray Crystallography 101

4 5 10

Methods – Structural Virology - 3D Tools

Cellular/Host interactions with the Capsid - CryoEM

Methods

Samples obtained by Co-IP from infection or Complexing after purification

4D Tools – Interplay of Approaches

Biochemistry

Biophysical Methods

Glycan arrays - Gal

Immunology

Molecular Biology

Pathology

Virology

&

Homology ModelingData Phasing

Cryo-EM and Image Reconstruction – ~9.8Å

Sequence AlignmentsX-ray Crystallography – 2.8Å

Methods

FUNCTIONAL ANNOTATION

8

209-217724-736 (C)

DE loop

HI loop

Capsid Interior

Capsid Exterior

BID

G

C

FE

H

Conserved Core

Eight-Stranded β-barrel

GH-l2GH-l5

GH-l1GH-l4

GH-l3

~400-630A

Conserved α-helix

AAV Capsid VP Structure

VP1 Numbering, 1-208/216 – Not observed

AAV structure

Building the AAV Capsid – 60 VPs (~220-735)

5f1

5f2

5f3

5f4REF

5-fold

4980Å2

HI loop

REF

3f13f2

10250 Å2

REF

2F

2800 Å2

5-Fold Axis

3-Fold Axis

2-Fold Axis

AAV structure

Xie et al., 2002210

725

216

7343

736

218

AAV1 AAV4 AAV5 AAV6218

736

220

736

220

737

AAV7 AAV8 AAV9

735

220

AAV2

Xie et al., 2002

217

736

AAV3

Lerch et al., 2010

Family Portrait – 2.5 – 3.45 Åresolution

Govindasamy et al., 2006 Ng et al., 2010

DiMattia et al, 2010Nam et al., 2007

AAV structure

9

INSIDE

5f

3f3f2f

5f

3f3f2f

5f

3f3f2f

DE loop

OUTSIDE

AAV4AAV2 AAV5

Conserved and Variable Features

Hypothesize that serotype specific phenotypes

receptor attachment, transduction efficiency, and

antibody recognition are likely facilitated by variations in

analogous capsid surface regions

AAV structure

EXTERIOR

INTERIOR

N

C

IX

IVIII IV

VII

III

II

V

VI

Variable regions I-IX (Cα > 1.0Å)

Superimposed Structures – Identifies common Variable Regions

Comparison of AAV1-AAV9

αA

AAV structure

EXTERIOR

N

C

Superimposed Structures – Identifies common Variable Regions

Comparison of AAV1-AAV9

αAVRI, VRII, VRIII, VRIV, VRVVRVI, VRVII, VRVIII, VRIX

5-fold pore

What role do these VRs play in receptor attachment, transduction efficiency, and antigenic diversity?

AAV structure

10

Capsid surface variations dictated serotype specificity

Genome Packaging – HI loopDiPrimio et al., 2008

TransductionShen et al., 2007Asokan et al., 2010Pulicherla et al., 2011

Receptor BindingOpie et al., 2003Kern et al, 2003Wu et al, 2006Schmidt et al., 2008DiMattia et al, in prepExcoffon et al, 2008Bell et al., 2012

TraffickingNam et al, 2011Zhou et al, in prep

Genome PackagingBleker et al. 2005Sonntag et al., 20062008

TransductionWu et al., 2000Lochrie et al., 2006Shen et al., 2007Li et al., 2012

The Multifunctional AAV Capsid

TransductionLi et al., 2008

Kotchey et al., 2011Pulicherla et

al., 2011

AntigenictyWobus et al., 2000Lochrie et al., 2006Gurda et al., 2012

McGraw et al., 2012

AAV VRs and Example Functional Roles

AAV function

Examples of Rational Engineering

AAV2.8 – Chimera between AAV2 and AAV8

Congenital heart failure and other cardiomyopathies

AAV2.5 – Chimera between AAV2 and AAV1

Muscular Dystrophy

AAV2-Y/F– Change of Surface tyr to phe

Hemophilia and other disease targets

11

Example 1 – AAV2i8

IX

IVIIIEXTERIOR

INTERIOR

N

IV

VII

C

III

II

V

VI

Variable regions I-IX (Cα > 1.0Å)

AAV2 HS binding Region and AAV5 Sialic Acid Binding Region:

ADK8 binding site in AAV8

Asokan et al.

Nature Biotech., 2010

Chimeras of AAV2 585-RGNRQA-590, contains

R585 and R588, with corresponding amino acids

from AAV1, AAV3, AAV4, AAV5, AAV7-AAV9

Re-engineering

RGNRQA QQNTAP QQNTAP QANTGP QTNTGP QTNGAP NATTAP

Dose

1 × 1011 vg

CBA-luc

AAV2 AAV8 2i8 2i10 2irh38 2irh2 2irh11

Luciferase transgene expression profiles in BALB/c miceDose 1 × 1010 vg, CMV-luc, in tail vien

AAV22i1 2i3 2i52i4 2i7 2i8 2i9

SSSTDP RGNRQA SSNTAP SNSNLP SSTTAP AANTAA QQNTAP SAQAQA

Re-Engineering

AAV2i8

Reengineering a receptor footprint of adeno-associated virus enables selective

and systemic gene transfer to muscle

Asokan et al., 2010, Nature Biotech

Re-Engineering

Protein Levels – 2 weeks Genome Levels – 2 weeks

Protein Levels – 2 weeks

AAv2i8

Chimera has prolonged circulation in blood

12

Re-Engineering

AAV2i8

• Can alter tissue tropism by mutating VR regions

•The chimera likely interacts with a receptor(s) distinct

from those used by AAV2 and AAV8.

• Or the increased circulation half-life of AAV2i8 allows

sequestration in tissues other than the liver through

heparan sulfate–independent uptake mechanisms.

•Chimera is antigenically distinct from AAV2

•Ongoing structure and functional studies

What do we know so far for this chimera?

Example 2 – AAV2 Y to F mutants

• Inhibition of Epidermal growth factor receptor protein tyrosine kinase

(EGFR-PTK) signaling decreases ubiquination of AAV2 capsids.

• Ubiquination of AAV2 capsid proteins tags them for proteasome degradation

• Mutations of capsid surface exposed tyr residues should inhibit

phosphorylation and ubiquitination and thus degradation of AAV vectors

by the cytoplasmic proteasome

• Strategy

• Identify Tyr residues on AAV2 Capsid Surface (also Ser and Thr residues)

• Mutate to Phe and test transduction phenotype in vitro and in vivo

Zhong et al., Molecular Therapy, 2007

Collaboration with Arun Srivastava and Sergei Zolotukhin

Re-engineering

2-fold 3f1

3f2

5f1

5f2

5f3

5f4

3f

Y252Y272

Y704

Y730

Y700

F444

F500

ref

R484

R588

R585R487K532

AAV2Heparan Residues : blue

AAV2-PHE residues : monomer type

AAV2 – Y to F

252, 272, 444, 500, 700, 704, 730

Re-engineering

13

2,000 vg/cell; 48 hrs

Transduction efficiency of wt and surface-exposed tyrosine residue

mutant capsid scAAV2 vectors in HeLa cells

Mock scAAV2-EGFP Y-F500

Y-F252 Y-F272 Y-F704

Y-F700 Y-F444 Y-F730

Tra

nsg

en

e e

xp

ressio

n

(Pix

el2

/vis

ual fi

eld

×10

4)

Mo

ck

scA

AV

2-E

GF

P

Y-F

500

* P<0.01 vs scAAV2-EGFP; ** P<0.001 vs scAAV2-EGFP

0

10

20

30

40

50

60

70

80

* *

**

**

**

**

Y-F

252

Y-F

272

Y-F

704

Y-F

700

Y-F

730

Y-F

444

Re-engineering

50X

Lobes

Transduction efficiency of wt and surface-exposed tyrosine residue

mutant capsid scAAV2 vectors in murine hepatocytes in vivo

Y-F272 Y-F444 Y-F500


Y-F700 Y-F704 Y-F730

1x1010 vg/mouse

Y-F272 Y-F444 Y-F500


Y-F700 Y-F704 Y-F730

1-week 2-weeks

Y730 F 3-log Increase in transduction

Re-engineering

Example 3 - AAV2.5

I. Differ between AAV2 and AAV3b and muscle transducing AAV serotypes

II. Located in a structurally variable region (VR, as defined by Govindasamy et al.)

III.Located in an AAV2 antigenic region that recognizes an antibody for which there is

no cross-reactivity from the muscle transducing serotypes , e.g. A20

Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational

Optimized AAV Vector (Bowles et al, Mol. Ther. 2011)

Challenges to Address:• Low/Transcient Transduction by AAV2

• Neutralization by pre-existing capsid antibodies

Criteria for Residue Selection for Rational Mutagenesis:

Bench to Bedside

14

AAV2.5

1

4

5

2

Lung

Brain

Retina

Retina

Brain

Broad

Muscle

Muscular

Dystrophy

Emphysema

Cystic Fibrosis

CNS diseases

Blindness

Emphysema

Diabetes

Blindness

Blindness 8

Muscle

Liver

Muscle

Liver

Muscle

Muscular

Dystrophy

Hemophilia

Diabetes

Hemophilia

Heart Disease

Liver

Hemophilia

Diabetes

Heart Failure

6

7

9



Tran

sgen

e

2

2

9875

2

2

1

9

9

9

8

8

8

7

7

7

5

5

1

1

1

Bench to Bedside

229 342240 250 260 270 280 290 300 310 320 330(229)

WHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQS--GASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTaav2 cap(228)

WHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQS--GASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVRGVTQNDGTTTIANNLTSTaav3a cap(228)

WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSAST-GASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTaav1 cap(228)

WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAG-STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTaav7cap(229)

WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTaav8cap(229)

WHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTaav9 cap(228)

343 456350 360 370 380 390 400 410 420 430 440(343)

VQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTN-TPSaav2 cap(340)

VQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSaav3a cap(340)

VQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQ-NQSaav1 cap(341)

IQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGaav7cap(342)

IQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTT-Gaav8cap(343)

VQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTI-NGSaav9 cap(342)

456 569470 480 490 500 510 520 530 540 550(456)

SGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIaav2 cap(452)

SGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIaav3a cap(453)

SGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIaav1 cap(453)

GGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFG-KTGATNKTTLENVLMTNEEEIaav7cap(455)

GGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIaav8cap(455)

SGQNQQ-TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIaav9 cap(454)

570 683580 590 600 610 620 630 640 650 660 670(570)

RTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSaav2 cap(566)

RTTNPVATEQYGTVANNLQSSNTAPTTGTVNHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSaav3a cap(567)

KATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSaav1 cap(567)

RPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSaav7cap(568)

KTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSaav8cap(569)

KTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSaav9 cap(567)

627 740640 650 660 670 680 690 700 710 720 730(627)

HTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL-aav2 cap(623)

HTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL-aav3a cap(624)

HTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL-aav1 cap(624)

HTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL-aav7cap(625)

HTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL-aav8cap(626)

HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL-aav9 cap(624)(Bowles et al, 2011)

**

**

VR I, II and IV and IX – Most Variable between these serotypes

I II

IV

IV

IX

AAV2

AAV3b

AAV1

AAV7

AAV8

AAV9

AAV2

AAV3b

AAV1

AAV7

AAV8

AAV9

AAV2

AAV3b

AAV1

AAV7

AAV8

AAV9

AAV2

AAV3b

AAV1

AAV7

AAV8

AAV9

AAV2

AAV3b

AAV1

AAV7

AAV8

AAV9

AAV2.5Structurally Ordered VP Region

*

Bench to Bedside

AAV2.53 Mutations Made – AAV1 amino acids Switched into AAV2

1. VR-II

AAV2- Q325T/T329V

2. VR-IV

AAV2-T450N/Q457N

3. VR-I & VR-IX (AAV2.5)

AAV2-Q263A, N705A, V708A,

T716N, T265 insertion

Bench to Bedside

3f2f

5f

3f

II

5f1-I,III 3f-IVIX

I,III3f1-IX2f-IV

II

3f

2f

5f

3f

IX

I

VII

I

N

IV

VI

I

C

III

II

V

VI

15

Virus Physical

Titers

Genome

Containing

Particle/ul

Heparin

Binding

Skeletal

muscle

Transduction

Hela Cos1 293

Parental

serotype

AAV2 1.3E+08 –

8.5E +08

+ + ++++ ++++ ++++

AAV1 1.3E+ 08-

1.5E+09

- ++++ + + +

Variants AAV2.5 5.0 E +08-

9.2E+09

+ +++ ++++ ++ ++

Q325T/T3

29V

6.9 E +08-

1.3E+09

N/D + N/D ++++ ++++

T450N/Q

457N

4.4 E +08-

9.3E+08

N/D ++ N/D ++++ ++++

WT and Mutant Variant PropertiesBench to Bedside

AAV2.5

T265

A706

A709

N717A263N

C

VP Monomer

A706

A709

N717

A263

T265

75º

Ref

Ref

3f2

2f

2f

5f

5fA263A265

A706

A709N717

VP Pentamer

A263A265

A706

A709

N717

T265-5f

Ref

5f

A263-5f

AAV2 A20

Antibody Epitope;

Transduction region

Bench to Bedside

C

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

3 7 21 28 42

Days post injection

RL

U/R

OI

2.5

aav1

aav2

AAV1 AAV2.5 AAV2

4 Days

Post injection

AAV2.5 Transduction

Intramuscular

Injection

AAV2.5

~5X

Higher than AAV2

VR-I & VR-IX - AAV2-Q263A, T265 insertion, N705A, V708A, T716N, T265 insertion

Bench to Bedside

16

• AAV2.5 shares only 10% antigenic cross-reactivity with AAV2 and AAV1 Abs

• Human Clinical Trials: 4/6 patients express clinical levels of mini-dystrophin gene

Sera AAV2 AAV2.5 AAV1

AAV2 1000 200 0

AAV2.5 200 800 100

Vecto

rs

AAV1 0 200 1000

Dilutions reducing

transduction by 50%

AAV2.5 Neutralization and Pre-Clinical DataBench to Bedside

• Second round of injection is possible with AAV2.5 after 1st injection with AAV2-AAT

B C

Low Dose 5x109 High Dose 1x1010 High Dose 5x1010 Control 1x1010

AAV2.5=right leg; AAV2=left leg

DA

Overall SUMMARY

Comparative structural studies of serotypes identify common variable surface loops that play a role in receptor recognition transduction, antigenicity

Chimeric capsids - new tropisms and antigenicity

Serotype specific phenotypes are facilitated by

variations in analogous capsid regions

AAV capsids likely utilization common AAV capsid

regions for receptor interactions, transduction, and antibody recognition

Information applicable for engineering cell/tissue targeted

tropism and antigenic variants

Documents

Structural biology for the optimization of gene …oge.med.ufl.edu/courses/gms 6001/IDP_MAM_9_18_2012.pdf · Structural biology for the optimization ... Structural biology for the