Berman, C.M.

Butty, V.L.

Doan, N.D.

Hendel, S.J.

Hoeben, R.C.

Kumar, C.M.

Moore, C.L.

Papa, L.J.

Shoulders, M.D.

Suen, P.H.

Uil, T.G.

Weickhardt, A.F.

The
discovery and optimization of biomolecules that reliably function
in metazoan cells is imperative for both the study of basic biology
and the treatment of disease. We describe the development, characterization,
and proof-of-concept application of a platform for directed evolution
of diverse biomolecules of interest (BOIs) directly in human cells.
The platform relies on a custom-designed adenovirus variant lacking
multiple genes, including the essential DNA polymerase and protease
genes, features that allow us to evolve BOIs encoded by genes as large
as 7 kb while attaining the mutation rates and enforcing the selection
pressure required for successful directed evolution. High mutagenesis
rates are continuously attained by trans-complementation of a newly
engineered, highly error-prone form of the adenoviral polymerase.
Selection pressure that couples desired BOI functions to adenoviral
propagation is achieved by linking the functionality of the encoded
BOI to the production of adenoviral protease activity by the human
cell. The dynamic range for directed evolution can be enhanced to
several orders of magnitude via application of a small-molecule adenoviral
protease inhibitor to modulate selection pressure during directed
evolution experiments. This platform makes it possible, in principle,
to evolve any biomolecule activity that can be coupled to adenoviral
protease expression or activation by simply serially passaging adenoviral
populations carrying the BOI. As proof-of-concept, we use the platform
to evolve, directly in the human cell environment, several transcription
factor variants that maintain high levels of function while gaining
resistance to a small-molecule inhibitor. We anticipate that this
platform will substantially expand the repertoire of biomolecules
that can be reliably and robustly engineered for both research and
therapeutic applications in metazoan systems

Chet M. Berman (6083822)

Louis J. Papa (1545424)

Samuel J. Hendel (6083825)

Christopher L. Moore (4919812)

Patreece H. Suen (6083828)

Alexander F. Weickhardt (6083831)

Ngoc-Duc Doan (1784485)

Caiden M. Kumar (6083834)

Taco G. Uil (2584453)

Vincent L. Butty (449351)

Robert C. Hoeben (6083837)

Matthew D. Shoulders (1323849)

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An Adaptable
Platform for Directed Evolution in Human
Cells

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An Adaptable Platform for Directed Evolution in Human Cells

Leiden University Scholary Publications

https://figshare.com/articles/An_Adaptable_Platform_for_Directed_Evolution_in_Human_Cells/7464695

An Adaptable Platform for Directed Evolution in Human Cells

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NARCIS

Leiden University Scholary Publications