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Synthetic dosage-compensating miRNA circuits allow precision gene therapy for Rett syndrome
Michael J. Flynn
1*
, Acacia M.H. Mayfield
2*
, Rongrong Du
2
, Viviana Gradinaru
2†
, Michael B. Elowitz
1,2,3,†
1
Division of Engineering and Applied Sciences, California Institute of Technology, Pasadena, CA 91125
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125
3
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125
* These authors contributed equally
Correspondence:
melowitz@caltech.edu
and viviana@caltech.edu
Abstract
A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight
therapeutic window. For example, loss of
MECP2
function causes Rett syndrome, while its duplication
causes
MECP2
duplication syndrome. Viral gene delivery methods generate variable numbers of gene
copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we
introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of
Mecp2
expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett
syndrome gene therapy. Single molecule analysis of endogenous and ectopic
Mecp2
mRNA revealed
precise, sustained expression across a broad range of gene dosages. Delivered systemically in a
brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24
weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic
miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and
efficacy of gene therapy.
One sentence description:
A synthetic miRNA-based incoherent feed-forward loop circuit embedded
in a gene delivery vector overcomes the challenge of overexpression toxicity in a mouse model of Rett
syndrome gene therapy.
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Introduction
Gene therapy promises to enable lasting cures for genetic diseases by delivering corrected copies of
genes mutated within a patient. Recently, the field has achieved clinical successes addressing spinal
muscular atrophy (SMA) (
1
), hemophilia (
2
4
), and inherited retinal dystrophy (
5
), with other
applications in the pipeline (
6
). New technologies, such as adeno-associated viral vectors (AAVs) that
target specific cell types at high efficiency (
7
), may accelerate progress in the coming decade.
However, many gene therapies face a ‘Goldilocks’ problem: too little expression of the gene leads to
the disease phenotype, but too much expression can induce other disease phenotypes. For example,
loss of function of the
SYNGAP1
gene results in non-syndromic intellectual disability and epilepsy (
8
)
while overexpression of
SYNGAP1
leads to a pronounced depression of excitatory signaling in vitro (
9
).
Similarly,
UBE3A
deficiency causes Angelman’s syndrome, but duplication and triplications are
associated with autism spectrum disorder (
10
). Overexpression toxicity is also a concern in clinical
gene replacement. For example, loss of function mutations in
SMN1
cause spinal muscular atrophy, but
overexpression of
SMN1
through gene therapy led to clinically silent but concerning dorsal root
ganglion pathology in mouse and non-human primate studies of SMA gene therapy (
11
13
).
Rett syndrome presents the prototypical Goldilocks problem. It is a severe neurodevelopmental disease
caused by loss-of-function mutations in the gene encoding MeCP2, a methyl-CpG/A binding protein
(
14
). MeCP2 binds to methylated regions of the genome and serves as a binding hub and bridge to the
NCoR/SMRT co-repressor complex to repress methylated genes, an essential function for brain
maturation (
15
). Since
MECP2
is on the X chromosome, mutations in the gene lead to different
symptoms between males and females. Human males cannot survive with a single non-functional copy
of
MECP2
(
16
)
.
In human females heterozygous for a mutation in
MECP2
, Rett syndrome is
characterized by a severe developmental regression at 7-18 months of age, progressive loss of speech
and hand use, ataxia, and acquired microcephaly, among other symptoms (
17
). MeCP2-deficient male
mice show reductions in lifespan, brain size, neuron soma size, synapse counts, dendritic spine density,
and electrophysiological activity (
18
). However, mild overexpression of MeCP2 also leads to a disease
phenotype (
19
) and duplication of the
MECP2
gene causes another disorder,
MECP2
duplication
syndrome (
20
). In engineered mice, Cre-based reactivation of
Mecp2
expression from its endogenous
genomic context alleviates disease phenotypes, suggesting that the condition is reversible (
21
).
However, gene therapies based on AAV-mediated delivery of
Mecp2
to Rett model mice have induced
toxicity (
22
27
), specifically from overexpression of the MeCP2 protein (
23
). Thus, a critical
requirement for Rett syndrome gene therapy is to express MeCP2 within a narrow therapeutic window.
In this work, we designed a synthetic biological circuit that provides this capability, and demonstrated
that it can improve Rett syndrome gene therapy in vivo. This compact miRNA-based incoherent
feedforward loop (IFFL) circuit limited
Mecp2
expression and reduced its sensitivity to gene dosage in
cell culture. Further, it restricted ectopic
Mecp2
mRNA to levels comparable to, but not exceeding,
those of endogenous
Mecp2
in the mouse brain. Finally, a gene therapy vector containing the circuit
outperformed unregulated gene therapy, improving behavioral symptoms over a timescale of 24 weeks.
These results demonstrate that an integrated miRNA-based gene circuit can improve gene therapies
for Rett syndrome and likely for other genetic diseases as well.
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Results
Modeling predicts incoherent feedforward regulation can tune protein expression to within a
therapeutic window
Expression patterns of ectopic genes may deviate significantly from the endogenous distribution of
expression for several reasons. First, the number of gene copies delivered to an individual cell can vary
by orders of magnitude due to varying uptake efficiency of different organs and cell types (
Figure 1A
,
upper right) (
28
,
29
), spatial gradients around a direct injection site (
Figure 1A,
bottom left) and
Poisson-distributed stochastic vector uptake by individual cells (
Figure 1A,
bottom right) (
30
). Second,
engineered promoters typically used in gene therapy vectors often induce stronger expression than
endogenous promoters, potentially leading to toxicity from even a single copy (
Figure 1B,
left panel)
(
31
). Finally, for an X-linked disease such as Rett syndrome, cells may express either zero or one copy
of the endogenous gene due to X inactivation. Thus, even if an ectopic gene is expressed at the
physiological levels this could still lead to overexpression in cells that express the endogenous copy
(
Figure 1B
, right panel).
Current approaches to limiting ectopic expression include optimizing the promoter (
32
) or incorporation
of target sites for endogenous miRNAs (
33
). However, while these approaches generally reduce mean
expression relative to unregulated constructs, they cannot actively adapt to variation in gene dosage.
The incoherent feedforward loop (IFFL) is an adaptive biological circuit motif that could address these
challenges (
34
,
35
). Previous work has shown that synthetic IFFL circuits can successfully buffer gene
expression against variations in gene dosage (
36
39
), noise from upstream regulators (
40
),
competition for cellular resources (
39
,
41
), or general perturbations (
42
). Here, we consider IFFLs in
which a target gene and its negative regulator are co-transcribed, so that higher gene dosage leads to
greater transcription rates of both components (
Figure 1C
) (
34
,
35
,
43
). A simple mathematical model
of such an IFFL showed that, above a minimal expression level, the two effects can cancel out to
maintain a fixed mean expression level of the target gene across a wide range of gene dosages
(
Figure 1D
,
Methods
). We further extended this model to incorporate discrete gene copy numbers,
stochastic vector delivery, and bursty gene expression kinetics. We also incorporated a strong promoter
so there is overexpression at even a single copy. Simulated IFFLs operating in these regimes
successfully regulated the distribution of expression to be similar to a target endogenous distribution,
across different MOIs (
Figure 1E, Methods
). These results suggested that a suitably engineered IFFL
could generate a more robust gene therapy expression system.
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Figure 1. Mathematical modeling predicts that incoherent feedforward loop circuits can maintain gene
expression within a therapeutic window.
(A)
Gene therapy contends with multiple sources of variability in expression. Ideally, all cells would receive the
same number of viral genome copies, and express the correct amount of the therapeutic gene (upper left).
However, viral uptake rates can vary greatly by organ and cell type (upper right), such that a dose that is
therapeutic in one organ (e.g. brain, blue) may be toxic in another that takes up viral vectors at a higher rate
(e.g. liver, red). With direct injection, cells close to the injection site receive more copies than cells farther away
(lower left). Finally, even with correct mean delivery, viral uptake remains subject to stochastic variation (lower
right).
(B)
The high level of expression induced by synthetic promoters commonly used in gene therapy may cause
toxic overexpression from even a single transgene copy (left). Additionally, for X-linked genes like
MECP2
,
approximately half of cells in affected females express a fully functioning endogenous copy. The gene therapy
must not overexpress MeCP2 when its expression is added to the wildtype allele (right).
(C)
Schematic of an incoherent feedforward loop motif in which a therapeutic gene is co-expressed with its own
negative regulator.
(D)
Therapeutic gene expression as a function of gene dosage, as modeled for an idealized IFFL. The
increasingly negative action of the repressor (R, black) compensates for increases in gene dosage, leading to
regimes where large changes in gene dosage yield nearly the same output expression of the circuit (blue),
preventing overexpression.
(E)
Simulated distributions of therapeutic gene expression at different viral MOI, either unregulated (left) or
regulated by an IFFL (right), compared to a target endogenous expression distribution (blue). Simulations
incorporate stochastic viral uptake, bursty transcription, and stochastic enzyme kinetics as well as an offset
between single-copy expression and the endogenous level of a therapeutic gene. The IFFL circuit compensates
for these sources of variation.
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A synthetic host-targeting miRNA module enables dosage compensation of
Mecp2
expression
In some natural genes, intronic miRNAs downregulate expression of their host gene, forming an IFFL
within a single transcript (
44
46
). This circuit architecture has also been demonstrated synthetically
(
36
) and could have desirable properties for gene therapy, since intronic miRNA expression cassettes
are non-immunogenic and genetically compact. However, it has not been established if synthetic
miRNA-based IFFLs can match the expression level of an endogenous mRNA, or whether they can
improve the function of an AAV-based gene therapy.
To address these questions, we designed a set of miRNA-based dosage compensating IFFL constructs
(
Figure 2A
). We engineered a divergent promoter made up of the CMV enhancer flanked by the
MeP229 promoter (
22
) in the forward direction and an intron-free Ef1ɑ promoter in the reverse direction
(‘ECM promoter’). The forward promoter drives expression of a previously characterized MeCP2-EGFP
protein fusion to facilitate analysis of protein expression (
26
). The reverse promoter drives expression
of unregulated mRuby3 as an indicator of gene dosage.
To implement IFFL regulation, we incorporated a miRNA expression cassette in a synthetic intron (
47
)
within the 3’UTR of
Mecp2-EGFP
. This miRNA cassette is based on the strong and well-characterized
miR-E backbone (
48
), which generates a miRNA complementary to a 21-bp sequence derived from
Renilla luciferase,
which is orthogonal to the human genome. To compare two different strengths of
regulation, we inserted either 1 or 4 copies of the target sequence into the 3’UTR, upstream of the
miRNA-containing intron, to create “1x” and “4x” circuits (
Figure 2A
). We also constructed an
“unregulated” control construct lacking both the miRNA and its target sites. All 3 constructs were less
than 4300 base pairs in length, and thus small enough to be efficiently packaged inside an AAV.
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Figure 2. Synthetic miRNA IFFLs can adapt to variations in gene dosage in cell culture.
(A)
Designs of 3 constructs based on a divergent promoter producing MeCP2-EGFP in the forward direction
and the mRuby3 dosage indicator in the reverse. The first circuit, labeled “unregulated”, has no miRNA targets
and no miRNA cassette and serves as an unregulated control. The second (1x) and third (4x) circuits contain a
miRNA cassette located within a synthetic intron in the 3’ UTR of
Mecp2-EGFP
, which respectively targets 1 or
4 fully complementary miRNA target sites upstream of the intron. All constructs are less than 4300 bp and fit
inside an AAV.
(B)
Workflow to characterize circuit performance at both the mRNA and protein levels. For protein, U2OS cells
were transiently transfected, cultured for 48 hours, and had protein expression measured by flow cytometry
(upper path). For mRNA, U2OS cells were transiently transfected, incubated for 48 hours, fixed, and then
analyzed with smFISH and confocal microscopy (lower path).
(C)
MeCP2-EGFP protein fluorescence as a function of mRuby3 dosage indicator for the 3 constructs, as
measured by flow cytometry. MeCP2-EGFP was proportional to dosage for the unregulated construct (gray), as
expected. For the 1x construct (medium blue), the slope was reduced, indicating a decreased responsiveness
to dosage. For the 4x construct (dark blue), MeCP2-EGFP expression was nearly independent of dosage over
2.5 orders of magnitude variation in gene dosage (framed region). This stable expression level was
approximately 3-fold above the fluorescence of untransfected cells (dashed black line). Here, and in D, shaded
regions represent ±1 standard deviation of the logarithmic expression values.
(D)
Mecp2-EGFP
transcript count as a function of average mRuby3 fluorescence, as measured by smFISH and
confocal microscopy. The relationship between
Mecp2-EGFP
transcripts and dosage indicator fluorescence
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largely agreed with the protein-level results for each construct. The 4x construct produced an expression level
that varied less than 4-fold over a greater than 300-fold range of dosage (framed region).
(E)
smFISH imaging of ectopic
Mecp2-EGFP
transcripts (upper row) and protein (middle row), as well as
mRuby dosage indicator (lower row). Cells displayed comparable levels of mRuby protein in all conditions
(bottom row), while
Mecp2-EGFP
expression decreases with stronger IFFL regulation at both transcript (upper)
and protein (middle) levels.
To test the ability of these circuits to compensate for variation in gene dosage, we quantified their
expression as a function of dosage at the protein and mRNA levels. For protein-level quantification, we
transiently transfected U2OS cells and analyzed MeCP2 expression using flow cytometry (
Figure 2B
,
upper path,
Methods
). Unregulated MeCP2 was expressed at a level proportional to gene dosage, as
expected (
Figure 2C
). The 1x construct reduced MeCP2-EGFP expression and its dependence on
dosage (slope of EGFP versus mRuby3) (
Figure 2C
). While it did not achieve complete dosage
compensation, it provided a useful intermediate-regulation condition for subsequent studies. The 4x
circuit generated behavior closer to that expected from the simplified IFFL model (
Figure 1D
), with
relatively constant (<3-fold variation) expression across a broad range (>300-fold) of gene dosage
(Figure 2C
).
To quantify these differences in expression at the mRNA level, we transiently transfected U2OS cells
with each construct, performed smFISH (
49
) against the
Mecp2-EGFP
transcript, and imaged both
protein fluorescence and transcripts using confocal microscopy (
Figure 2B,
lower path,
Methods
).
Consistent with the flow cytometry results, among cells expressing similar levels of the mRuby dosage
reporter, MeCP2-EGFP mRNA and protein expression levels decreased with increasing number of
miRNA target sites (
Figure 2E
).
To quantitatively measure the relationship between gene dosage and target mRNA levels, we
computationally segmented cells in the smFISH images and counted individual transcripts (dots in
Figure 2E
,
Methods
). The unregulated and 1x constructs showed linear and sublinear, but still
increasing, dependence on the mRuby3 dosage indicator, respectively (
Figure 2D
). The 4x construct
exhibited the lowest level of expression, which also varied less than 4-fold over a greater than 300-fold
range of dosage (
Figure 2D
).
To test for potential off-target effects of the miRNA regulation, we performed bulk-RNAseq and
compared transcriptome expression of a BFP-miRNA (miR-E) cassette to a negative control
transfection of a BFP-only expression vector (
Methods
). Few genes were relatively up- or
down-regulated in the miRNA condition and none contained partial sequence matches to the miRNA
(
Supplementary Figure 1
). These results suggest any sequence-specific perturbations from the
synthetic miRNA itself were minimal.
Taken together, these results indicate that the 4x IFFL circuit can establish dosage-insensitive
expression and reduce the magnitude of cell-cell variation in gene expression in vitro, without
perturbing endogenous gene expression.
AAV-delivered IFFL circuits reduce
Mecp2
mRNA to endogenous levels in mouse brains
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