RESUMO
Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing, and stimulus responses, which collectively define the thousands of unique cell types in the body. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for an intended purpose has arisen naturally through evolution. Here, we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell type specificity. We leverage innovations in deep neural network modeling of CRE activity across three cell types, efficient in silico optimization, and massively parallel reporter assays (MPRAs) to design and empirically test thousands of CREs. Through in vitro and in vivo validation, we show that synthetic sequences outperform natural sequences from the human genome in driving cell type-specific expression. Synthetic sequences leverage unique sequence syntax to promote activity in the on-target cell type and simultaneously reduce activity in off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs and demonstrate the required literacy to write regulatory code that is fit-for-purpose in vivo across vertebrates.
RESUMO
Embryonic stem (ES) cells can be differentiated into many neural cell types that hold great potential as cell replacement therapies following spinal cord injury (SCI). Coupling stem cell transplantation with biomaterial scaffolds can produce a unified combination therapy with several potential advantages including enhanced cell survival, greater transplant retention, reduced scarring, and improved integration at the transplant/host interface. Undesired cell types, however, are commonly present in ES-cell derived cultures due to the limited efficiency of most ES cell induction protocols. Heterogeneous cell populations can confound the interaction between the biomaterial and specific neural populations leading to undesired outcomes. In particular, biomaterials scaffolds may enhance tumor formation by promoting survival and proliferation of undifferentiated ES cells that can persist after induction. Methods for purification of specific ES cell-derived neural populations are necessary to recognize the full potential of combination therapies involving biomaterials and ES cell-derived neural populations. We previously developed a method for enriching ES cell-derived progenitor motor neurons (pMNs) induced from mouse ES cells via antibiotic selection and showed that the enriched cell populations are depleted of pluripotent stem cells. In this study, we demonstrate the survival and differentiation of enriched pMNs within three dimensional (3D) fibrin scaffolds in vitro and when transplanted into a sub-acute dorsal hemisection model of SCI into neurons, oligodendrocytes and astrocytes.
