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- EMDB-19412: hCD8_Darpin-AAV2 -

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Basic information

Entry
Database: EMDB / ID: EMD-19412
TitlehCD8_Darpin-AAV2
Map dataCryo-EM map of huCD8-AVV2
Sample
  • Virus: AAV expression vector pTR-UF50-BC (others)
KeywordsAAV2 with Darpins / VIRUS
Biological speciesAAV expression vector pTR-UF50-BC (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsChlanda P / Buchholz CJ
Funding support Germany, 1 items
OrganizationGrant numberCountry
Other privateChica and Heinz Schaller Foundation Germany
CitationJournal: Mol Ther / Year: 2024
Title: T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.
Authors: Muhammed Burak Demircan / Luca J Zinser / Alexander Michels / Mar Guaza-Lasheras / Fabian John / Johanna M Gorol / Samuel A Theuerkauf / Dorothee M Günther / Dirk Grimm / Florian R Greten / ...Authors: Muhammed Burak Demircan / Luca J Zinser / Alexander Michels / Mar Guaza-Lasheras / Fabian John / Johanna M Gorol / Samuel A Theuerkauf / Dorothee M Günther / Dirk Grimm / Florian R Greten / Petr Chlanda / Frederic B Thalheimer / Christian J Buchholz /
Abstract: One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs ...One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.
History
DepositionJan 16, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19412.png

Downloads & links

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Map

FileDownload / File: emd_19412.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of huCD8-AVV2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.69 Å/pix.
x 512 pix.
= 353.28 Å		0.69 Å/pix.
x 512 pix.
= 353.28 Å		0.69 Å/pix.
x 512 pix.
= 353.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.69 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00691
Minimum - Maximum-0.005601464 - 0.01637228
Average (Standard dev.)0.00056435494 (±0.0018874897)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 353.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM map of huCD8-AVV2 half

Fileemd_19412_half_map_1.map
AnnotationCryo-EM map of huCD8-AVV2 half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM map of huCD8-AVV2 half

Fileemd_19412_half_map_2.map
AnnotationCryo-EM map of huCD8-AVV2 half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AAV expression vector pTR-UF50-BC

EntireName: AAV expression vector pTR-UF50-BC (others)
Components
  • Virus: AAV expression vector pTR-UF50-BC (others)

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Supramolecule #1: AAV expression vector pTR-UF50-BC

SupramoleculeName: AAV expression vector pTR-UF50-BC / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 1463588 / Sci species name: AAV expression vector pTR-UF50-BC / Sci species strain: AAV2 / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Virus shellShell ID: 1 / T number (triangulation number): 1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 9492
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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