10.21433/VP9G-HD64
Byrne, Leah
University of California Berkeley
Day, Timothy
University of California Berkeley
Visel, Meike
University of California Berkeley
Dalkara, Deniz
Institut de la Vision
Dufour, Valerie
University of Pennsylvania
Pompeo Marinho, Felipe
University of Pennsylvania
Merigan, William
University of Rochester
Aguirre, Gustavo
University of Pennsylvania
Beltran, William
University of Pennsylvania
Schaffer, David
University of California Berkeley
Flannery, John
University of California Berkeley
Directed Evolution of AAV for Efficient Gene Delivery to Canine and
Primate Retina - Raw counts of variants from deep sequencing
UC Berkeley
2018
2018-12-25T08:00:00Z
en
dataset
180261221 bytes
1
Creative Commons Attribution 4.0 International (CC BY 4.0)
Efficient AAV-mediated gene delivery remains a significant obstacle to
effective retinal gene therapies. Here, we apply the process of directed
evolution – guided by deep sequencing and followed by direct in vivo
secondary selection of high-performing vectors with a GFP-barcoded library
– to create AAV viral capsids with new capabilities to deliver genes to
the outer retina in large animals. The resulting vectors resulted in
efficient targeting of photoreceptors, bipolar cells, and RPE cells in dog
retina and substantially increased efficiency of gene delivery to primate
outer retina. In addition, direct comparison of gene delivery across
animals revealed emergent species specificities in vector performance.
These new viral vectors will enable long-term and pan-retinal gene
therapies targeting outer retina cell types in large preclinical animal
models and establish deep sequencing-guided directed evolution as a
powerful approach for developing AAV vectors specialized for a multitude
of physical barriers and cellular targets.
Highly diverse (~1E+7) libraries of AAV variants were packaged such that
each virus contained a genome encoding its own capsid. Libraries were
pooled and injected intravitreally in canines or primates. After AAV
infection had occurred, retinal tissue and RPE cells were collected, and
cap gene variants were PCR amplified recloned, and repackaged for the
subsequent round of injection. Five rounds of selection were performed,
and error prone PCR was performed after the third round to introduce
additional diversity into the library. Following the selections, each pool
was subjected to deep sequencing in order to analyze the dynamics of each
individual variant and overall convergence of the library. Based on their
increase in representation relative to the original library, individual
variant capsids were chosen. Files contain raw data counts from deep
sequencing of AAV2-7mer libraries (in canine RPE and primate ONL) and
LoopSwap~588 library (in primate ONL).