Article
Engineering RNA export for measurement and
manipulation of living cells
Graphical abstract
Highlights
d
RNA exporters package and secrete RNA within protective
nanoparticles
d
Engineered exporters achieve high specificity for cargo RNA
d
Sequencing exported RNA barcodes enables monitoring of
cell population dynamics
d
Exporters enable cell-to-cell delivery and expression of
mRNA cargo
Authors
Felix Horns, Joe A. Martinez,
Chengcheng Fan, ..., Pamela J. Bjorkman,
Carlos Lois, Michael B. Elowitz
Correspondence
felix@caltech.edu (F.H.),
melowitz@caltech.edu (M.B.E.)
In brief
The development of RNA exporters,
based on capsids and nanocages, for
packaging and secretion of RNA, non-
destructive monitoring of cells, and cell-
to-cell delivery of mRNA.
Horns et al., 2023, Cell
186
, 3642–3658
August 17, 2023
ª
2023 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.cell.2023.06.013
ll
Article
Engineering RNA export for measurement
and manipulation of living cells
Felix Horns,
1,2,
*
Joe A. Martinez,
1
Chengcheng Fan,
1
Mehernaz Haque,
1
James M. Linton,
1
Victoria Tobin,
1
Leah Santat,
1,2
Ailiena O. Maggiolo,
3
Pamela J. Bjorkman,
1
Carlos Lois,
1
and Michael B. Elowitz
1,2,4,
*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
2
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA
3
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
4
Lead contact
*Correspondence:
felix@caltech.edu
(F.H.),
melowitz@caltech.edu
(M.B.E.)
https://doi.org/10.1016/j.cell.2023.06.013
SUMMARY
A system for programmable export of RNA molecules from living cells would enable both non-destructive
monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other
cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package
and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and
sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal res-
olution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression,
and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled
output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement
of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery
of RNA.
INTRODUCTION
As a central information carrier in the cell, RNA provides a power-
ful interface for reading and writing cell behaviors. Sequencing
RNA enables the readout of cell states. In parallel, expression
of RNA controls cell states. However, RNA is typically confined
within the cell that produced it, limiting its utility for molecular
analysis and intercellular communication. By contrast, the ability
to programmably export RNA molecules from cells could unlock
ways to both analyze and control living cells.
RNA export enables the non-destructive measurement of cell
dynamics. Single-cell RNA sequencing and hybridization-based
assays have revolutionized biomedicine by enabling researchers
to decipher the molecular types and states of individual cells at
genome scale.
1–4
However, physically accessing RNA for anal-
ysis generally requires lysis or fixation of cells, preventing one
from tracking the dynamic behavior of individual living cells
over time. Cell-free RNA is naturally secreted by cells in extracel-
lular vesicles (EVs) or upon cell death, and sequencing this
RNA can non-destructively reveal biomarkers of health and dis-
ease.
5–7
However, the low rates of natural RNA secretion
8
,
9
limit
the sensitivity and information content of cell-free RNA assays.
As an alternative approach, engineering cells to efficiently export
RNA molecules that encode information about cell populations
and states, then collecting and sequencing this exported RNA
could enable non-destructive measurement of cell dynamics
with enhanced sensitivity and information content compared
with natural cell-free RNA assays (
Figure 1
A).
RNA export also unlocks ways to manipulate cell behaviors.
The ability of RNA to encode proteins and regulate gene expres-
sion promises programmable control of cell behaviors. However,
therapeutic use of this capability remains limited by challenges in
delivering RNA to specific cell populations within tissues.
10
The
ability to engineer cells to export RNA raises the possibility of
creating therapeutic ‘‘delivery cells’’ that home to tissues, recog-
nize target cells, and locally deliver RNA circuits that execute
diverse functions within recipient cells, including altering their
gene expression, reprogramming cell fate, or selectively killing
cells in diseased states
11
(
Figure 1
A). This strategy could circum-
vent difficulties encountered with other delivery vectors in
achieving tissue and target specificity because cells are capable
of penetrating tissues and utilizing cell-based sensing and logic
to conditionally regulate localized RNA delivery. A foundational
component of this vision is a system that efficiently exports
RNA cargo within a vehicle that permits uptake and expression
of the RNA by non-engineered receiver cells.
Virus-like particles (VLPs) and EVs are attractive platforms for
export and delivery of RNA. Viral structural proteins, which form
capsids, and their natural interactions with RNA packaging sig-
nals (PSs) have been used to package and transfer RNA be-
tween cells in VLPs.
12–15
However, these approaches have
often relied on retroviral capsid proteins, such as those of
ll
OPEN ACCESS
3642
Cell
186
, 3642–3658, August 17, 2023
ª
2023 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
).