structural biology for the optimization of gene …oge.med.ufl.edu/courses/gms...
TRANSCRIPT
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)
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
AAV Serotypes and Gene Delivery
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
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
AAV Serotypes and Gene Delivery
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
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
AAV Serotypes and Gene Delivery
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
AAV Basics
Adapted From Gao et al., JV 2004
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
transduction efficiency
• 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
Adapted From Gao et al., JV 2004
Transduction Phenotypes
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
Mock scAAV2-EGFP Y-F252
Y-F700 Y-F704 Y-F730
1x1010 vg/mouse
Y-F272 Y-F444 Y-F500
Mock scAAV2-EGFP Y-F252
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
Adapted From Gao et al., JV 2004
Transduction Phenotypes
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