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<p>Technologies for engineering new functions into proteins are advancing biological
research, biotechnology, and medicine at an astounding rate. Building on fundamental
research of natural protein structure and function, scientists are identifying new
protein domains with previously undescribed properties and engineering new proteins
with expanded functionalities. Such tools are enabling the precise study of fundamental
aspects of cellular behavior and the development of a new class of gene therapies
that manipulate the expression of endogenous genes. The applications of these gene
regulation technologies include but are not limited to controlling cell fate decisions,
reprogramming cell lineage commitment, monitoring cellular states, and stimulating
expression of therapeutic factors. </p><p>While the field has come a long way in the
past 20 years, there are still many limitations. Historically, gene therapy and gene
replacement therapies have relied on over-expression of natural transcription factors
that activate specific endogenous gene networks. However, natural transcription factors
are often inadequate for generating efficient, fast, and homogenous cellular responses.
Furthermore, most natural transcription factors have complex structures and functions
that are difficult to improve or alter by rational design. This thesis presents three
novel and widely applicable methods for engineering transcription factors for programming
cell fate decisions in primary human cells. MyoD is the master transcription factor
defining the myogenic lineage. Expression of MyoD in certain non-myogenic lineages
induces a coordinated change in differentiation state. We use MyoD as a model for
developing our protein engineering techniques because myogenesis is a well-studied
pathway that is characterized by an easily detected change in phenotype from mono-nucleated
to multinucleated cells. Furthermore, efficient generation of myocytes in vitro presents
an attractive patient-specific method by which to treat muscle-wasting diseases such
as muscular dystrophy.</p><p>We first demonstrate that we can improve the ability
of MyoD to convert human dermal fibroblasts and human adipose-derived stem cells into
myocyte-like cells. By fusing potent modular activation domains to the MyoD protein,
we increased myogenic gene expression, myofiber formation, cell fusion, and global
reprogramming of the myogenic gene network. The engineered MyoD transcription factor
induced myogenisis in a little as ten days, a process that takes three or more weeks
with the natural MyoD protein. </p><p>While increasing the potency of transcriptional
activation is one mechanism by which to improve transcription factor function, there
are many other possible routes such as increasing DNA-binding affinity, increasing
protein stability, altering interactions with co-factors, or inducing post-translational
modifications. Endogenous regulatory pathways are complex, and it is difficult to
predict specific amino acid changes that will produce the desired outcome. Therefore,
we designed and implemented a high-throughput directed evolution system in mammalian
cells that allowed us to enrich for MyoD variants that are successful at inducing
expression of the myogenic gene network. Directed evolution presents a well-established
and currently unexplored approach for uncovering amino acid substitutions that improve
the intrinsic properties of transcription factors themselves without any prior knowledge.
After ten rounds of selection, we identified amino acid substitutions in MyoD that
increase expression of a subset of myogenic gene markers in primary human cells.</p><p>Rather
than guide cell fate decisions by expressing an exogenous factor, it may be beneficial
to activate expression of the endogenous gene locus. In comparison to delivering the
transcription factor cDNA, expression from the endogenous locus may induce chromatin
remodeling and activation of positive feedback loops to stimulate autologous expression
more quickly. Recent discoveries of the principles of protein-DNA interactions in
various species and systems has guided the development of methods for engineering
designer enzymes that can be targeted to any DNA target site. We make use of the RNA-guided
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to
induce expression of the endogenous MyoD gene in human induced pluripotent stem cells
(iPSCs). Through complementary base pairing, chimeric guide RNAs (gRNAs) direct a
Cas9 transcriptional activator to a target DNA sequence, leading to endogenous gene
expression. A current limitation of CRISPR/Cas9-based gene regulation is the potency
of transcriptional activation and delivery of the CRISPR/Cas9 components. To address
these limitations, we first developed a platform to express Cas9 and up to four gRNAs
from a single lentiviral vector. We then optimized the gRNAs and Cas9 transcriptional
activator to induce endogenous MyoD expression and differentiate iPSCs into myocyte-like
cells. </p><p>In summary, the objective of this work is to develop protein engineering
techniques to improve both natural and synthetic transcription factor function for
programming cell fate decisions in primary human cells. While we focus on myogenesis,
each method can be easily adapted to other transcription factors and gene networks.
Engineered transcription factors that induce fast and efficient remodeling of gene
networks have widespread applications in the fields of biotechnology and regenerative
medicine. Continuing to develop these tools for modulating gene expression will lead
to an expanded number of disease models and eventually the efficient generation of
patient-specific cellular therapies.</p>
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