(6s) Integrated Gene Circuit Design and Cellular Engineering: Probing and Reshaping the Genome to Control Cell Fate

The ultimate
goal of my research is to leverage synthetic biology to transform how we
understand cellular transitions and engineer cellular therapies.

I. Summary. Synthetic
biology aims to harness the power of biological systems to perform tasks such as
tumor surveillance, pathogen identification, and metabolite homeostasis. Native
biological circuits (e.g. connected networks of genes) query data within the cell to coordinate
cellular behaviors in space and time. Within mammalian systems, there exists an enormous
opportunity to use synthetic circuitry to dynamically access information in the
cell. As a chemical
engineer working in molecular systems biology, my research focuses on
integrating synthetic circuitry to interrogate and drive cellular behaviors.
As a postdoc, I identified topological stress across the genome as a primary
barrier to cellular reprogramming. This finding opens completely new questions
for how the structure of the human genome stabilizes cellular identity and
buffers cells against transitions to pathological states. Additionally, my
findings explain why particular circuit designs fail and suggest principles for
improving the performance of circuits integrated into the genome. I will establish a leading research
program focused on designing and constructing integrated synthetic circuits to
probe and actuate changes in DNA topology that drive changes in cell fate.

II. Research Accomplishments. Spanning a range of model organisms from
yeast to human cells, I have engineered systems for dynamic behaviors across
multiple scales from the molecular design of noncoding RNA devices to
optimization of large transcriptional networks.

Graduate:
Working with Dr. Christina Smolke at the California Institute of
Technology, I constructed synthetic gene circuits and developed a class of
genetic control systems called molecular network diverters. By interfacing
these molecular network diverters with the native MAPK signaling pathway, I
could temporally and spatially control cellular decision-making events (Galloway, K.E. et al. Science. 2013,). Beyond controlling cell
fate, my work highlighted
that integrated negative regulators can buffer a system against noise
amplification mediated through positive-feedback loops by providing a
resistance to amplification (Galloway, K.E. and Franco, E. Computational Methods in Synthetic Biology, 2015). By
constructing synthetic circuitry, I will continue to illuminate paradigms in
biological control and apply those principles to enhance cellular engineering.

Postdoc: As a postdoc at USC in
Dr. Justin Ichida’s laboratory, my work has focused on elucidating and overcoming the
reprogramming roadblocks to the robust generation of mature cell types.
Accurately modeling neurological disorders with in vitro cellular models relies on reliable methods to generate the
distinct neural subpopulations affected by the disease. When I began my
project, the conversion process was extremely inefficient, requiring
large-scale efforts to generate only a few hundred cells. Moreover, the central
mechanistic rules for direct lineage conversion were undefined. Today, as a
direct result of my work to improve the reprogramming process, I can robustly
generate thousands of cells with signatures of enhanced maturity (Babos,
K.N.*, Galloway, K.E.*, et al. Balancing hyperproliferation, transcription to
drive cellular reprogramming. In
Preparation.,). In addition to improving the reprogramming process, my work
uncovers a previously unrecognized explanation for why only rare cells undergo
transcription factor-mediated reprogramming successfully. I found that cellular
reprogramming is limited to a small population of cells equipped to process the
dual, competing demands of hyperproliferation and hypertranscription. High
rates of transcription and replication accelerate the rate of DNA tangling
(e.g. supercoiling). Only cells equipped with high expression of
topoisomerases, enzymes that relax DNA supercoils, are
capable of mediating the massive genomic and transcriptional realignment
to convert from one state to another. My findings suggest that topological
stress profoundly impacts the function of gene networks (e.g. native or
synthetic circuits). By more precisely defining the impact of topological stress on
gene circuits, I will elucidate principles for buffering (or alternatively,
harnessing) topological stress to enhance the performance and capabilities of
genome-integrated synthetic circuits.

III. Research Interests: Prokaryotic systems and
single-celled eukaryotic organisms have dominated the field of synthetic
biology and revealed important paradigms in cellular biology including the role
of feedback, noise, and cooperativity. While translation of synthetic biology
to mammalian systems has been slower, with the advent of improved genetic tools
for mammalian cells (e.g. gene therapy approaches including CRISPR
technologies, AAVs), synthetic circuits are positioned to massively reshape how
we study and treat diseases. Elucidating the principles of mammalian
circuit design offers the opportunity to engineer cellular behaviors and to
identify and target diseased states. Further, understanding the how cell
types differentially process classes of circuits will improve our ability to
predict the response of native transcriptional networks and design systems that
are optimally wired for their function and context.

Focus 1. Elucidating the
systems-level principles of cell fate transitions.

>Mapping
transcriptional states: Characterizing the influence of hyperproliferation on
the transcriptional trajectories between cell states.

>Proliferation as a
motif: Defining the molecular impact of proliferation on cellular transitions

>Measuring transition
speed: Characterizing the dynamics of proliferation-mediated transitions
between cell states.

Focus 2. Integrated gene circuit design

>Elucidating principles of three-dimensional circuit design.

>T.A.N.G.L.E.S. (Topologically-Affected Network of Genes Linking
Expression to State) as probes.

> Identifying robust designs for genome-integrated circuits: Mining
the mammalian genome for structural designs.

>Funding

Continuous independent funding as
postdoctoral fellow with acquisition of additional mini-grants

NIH
Ruth L. Kirschstein NRSA Postdoctoral Fellowship (Fall 2015 – Fall 2018)

California
Institute of Regenerative Medicine Postdoctoral Fellowship (Fall 2013 – Fall
2015)

Doerr USC Stem Cell Challenge Award
$10,000 project (2017)

Fluidigm USC Single Cell Project Grant
$9,000 in reagents and materials (June 2016)

>Publications

Galloway,
K.E.*, Babos, K.*, and  Ichida, J.I. Balancing hyperproliferation,
transcription to drive cellular reprogramming. (In preparation). *These authors
contributed equally to this work.

Galloway,
K.E., Babos, K., and  Ichida, J.I.
Enhancing in vitro disease models of
ALS through precise motor neuron subtype engineering. (In preparation).

Galloway,
K.E*., Yu, H. *., Segil, N. I., and 
Ichida, J.I. Building a motor neuron enhancer map using ATM-ChIPseq. (In
preparation). *These authors contributed equally to this work.

Ichida,J.I. Staats, K., Davis-Dusenbery,
B.N., Clement, K., Galloway, K.E.,
Babos, K.N. Son, E.Y., Kiskinis, E., Nicholas
Atwater, N. , Gu ,H, Gnirke, A., Alexander Meissner,
Kevin Eggan. Comparative genomic analysis of
embryonic, lineage-converted, and stem cell-derived motor neurons. Development.
(In resubmission).

Galloway, K.E. and  Ichida, J.I. 
Modeling neurodegenerative diseases and neurodevelopmental disorders
with reprogrammed cells. Stem Cells,
Tissue Engineering and Regenerative Medicine. D.A. Warburton, Ed. (World
Scientific, New Jersey, 2015).

Franco,
E., and Galloway,
K.E. Feedback loops in biological
networks. Computational Methods in
Synthetic Biology. M. A. Marchisio, Ed. (Springer New York, 2015), vol.
1244, pp. 193-214.

Galloway K.E., Franco, E., and Smolke, C.D.
Dynamically reshaping signaling networks to program cell fate via genetic
controllers. Science. 2013.
341:1235005.

Chen,
Y.Y*, Galloway, K.E.*, and Smolke,
C.D. Synthetic biology: advancing biological frontiers by building synthetic
systems. Genome Biology. 2012.
13:240.  * These authors contributed
equally to this work.

Kostal, J., Mulchandani,
A., Gropp, K.E., and Chen, W.  A. Temperature Responsive Biopolymer for
Mercury Remediation. Environmental
Science & Technology. 2003. 37, 4457-4462.

Teaching
Interests:

My comprehensive
Chemical Engineering education from UC Berkeley and the California Institute of
Technology has equipped me to teach classes in the core Chemical Engineering
curriculum (kinetics, transport, and thermodynamics) at both undergraduate and
graduate levels. I’m also very familiar with control theory and molecular and
cellular biology. The problem-solving skills I acquired as a chemical engineer
have set me up for success in elucidating the systems principles of biological
systems. Thus, I am enthusiastic to teach chemical engineering principles to
the next generation of students. Over my four years as a postdoc, I have
directly mentored 15 students (5 grad, 8 undergrad, 2
high school) which has motivated me to develop strategies for training my
students 1) to be effective in lab and 2) to develop their research and
presentation skills. 

Teaching
experience: In
addition to serving as a teaching assistant for two classes at Caltech, I
taught the freshman chemistry lab at Harvey Mudd
College (HMC) in spring 2013. HMC is highly regarded for its focus on teaching
and is currently ranked as the #2 engineering school without a doctorate
program. Since all HMC students are obligated to take the freshman chemistry
course, many students who were uncomfortable in a laboratory setting approached
the class with dread. In order to capture my students’
interest, I began each class by engaging them with a bigger picture view of the
purpose of the lab (e.g. why do we care about error propagation, synthesis
yields, carbonate chemistry, etc.) as well as a general overview of what to
expect. One student noted in the teaching reviews, “I thought the way Prof.
Galloway started each lab with a little PowerPoint overview (where she went
over the prelab if it was confusing, and just generally went over the process
(was good)).” Helping my students understand the bigger picture of the material
made the labs more meaningful and empowering. With this level of engagement, I
could challenge the students to think deeper when troubleshooting their
experiments and analyzing their results. Whenever a student asks a question, I
try to refrain from giving direct answers, instead offering another question
that leads them toward answering their initial question. Letting the students
discover the answers for themselves helps them to build a better understanding
and confidence in their deductive skills. Long-term, I want students to
recognize that asking good questions is fundamental to science, whether in
theory or in practice. One student reflected that I was “always available to
answer questions about lab procedure and write-up, and provides ample help
without any hand-holding.” Overall, my students were engaged and left with a
positive view of chemistry. I received excellent evaluations with scores of
either 6 or 7 (out of 7) in all categories as well as this gratifying comment:
“Prof Galloway is extremely supportive of her students and she clearly wants us
all to succeed, not only in lab, but in our careers as scientists.”

Philosophy: While at Caltech, I completed the Caltech
Project in Effective Teaching (CPET) certificate program, which introduces
teaching pedagogy and practices through six seminars. From CPET, I learned that
effective communication is the key to being an
excellent teacher. I have become an extremely effective communicator as
evidenced by the awards I have won for scientific communication, including the
Everhart Lecture Award and First Place at the Annual USC Postdoctoral
Symposium. In teaching, I have used techniques introduced to me through CPET
training including the Socratic Method, inductive teaching, and story-telling.
My goal is to help my students engage with concepts and approach a deeper
understanding of scientific principles. In particular, I
find that requiring students to draw out systems improves mechanistic
understanding, develops intuition for the way processes work, and reduces
experimental errors.