SCIENCE sciencemag.org
THERAPEUTICSGene therapy for pathologic
gene expression
group of [NiFe] hydrogenases is only dis-
tantly related to the H/Q-module of the com-
plex I superfamily. Thus, respiratory complex
I has evolved by combining ancient modules
from two different groups of hydrogenases
and Mrp sodium–proton antiporters. This
core structure was then retained all the way
from bacterial to mammalian complex I.
Several conclusions can be drawn from
the adaptive development of the super-
family originating from simple soluble hy-
drogenases and leading to the large and
complicated molecular machine of mam-
malian complex I. Proton pumping by the
PI/PP-module is driven by redox chemistry
catalyzed by the H/Q module, irrespective of
its orientation in the membrane domain. Dif-
ferent additional ion-translocating modules
from Mrp are docked onto this central unit.
In the PD-module, even two closely related
modules are stacked onto the PP-module. It
seems that this works because the central
axes of protonable residues common to all
modules of the Mrp transporter can connect
flexibly with each other. This is in line with
the proposed mechanism of electrostatic en-
ergy transmission into the central axis from
the H/Q-module and from one pump site to
the next, both of which feature broken trans-
membrane helices found in many ion trans-
porters. During the conversion from the H
into the Q-module, the hydrogen reactive
[NiFe] center was transformed into binding
pockets for different kinds of hydrophobic
quinones. Remarkably, the fold surrounding
this active site and even a substantial num-
ber of critical residues are highly conserved
( 9 ). This holds in particular for a cluster of
three critical loops that connect the active
site with the common membrane anchor,
which has been implicated in generating the
electrostatic pulse transmitted toward the
pump modules in the membrane domain of
complex I ( 4 , 11 ) and is already present in
membrane-bound hydrogenase ( 6 ).
Studying the conserved structural ele-
ments and understanding the functional
adaptions of complex I superfamily mem-
bers will greatly help to understand the still
poorly understood molecular mechanism of
these distinct redox-driven ion pumps. j
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10 .1 126 /science.aaw 0493Haploinsufficiency in disease can be overcome by boosting
gene expression with CRISPR
By Lindsey E. Montefiori and
Marcelo A. NobregaH
aploinsufficiency arises when one
copy of a gene is functionally lost, of-
ten through nonsense or frameshift
mutations or small chromosomal
deletions. The resulting monoallelic
expression is not sufficiently com-
pensated for by the intact allele, ultimately
leading to decreased expression of the gene
product and resulting in pathologic pheno-
types ( 1 ). What are the therapeutic options
for diseases rooted in insufficient gene ex-
pression? One possible viable option is to
restore normal gene expression levels by
enhancing their transcription in a targeted
fashion. On page 246 in this issue, Matharu et
al. ( 2 ) report a CRISPR-based gene-activation
approach that can increase the expression of
normal endogenous genes in a tissue-specific
manner, setting the stage for the develop-
ment of new gene-regulating therapies for
gene dosage–associated diseases.
Among the emerging applications of
CRISPR-based gene editing are techniques
that use a catalytically inactive Cas 9 en-
zyme (dCas 9 ) fused to a protein domain to
modulate transcription ( 3 ). These fusion
proteins can be recruited by way of guide
RNAs (gRNAs) to specific genomic locations,
including promoters and cis-regulatory ele-
ments such as enhancers, which regulate
gene expression. If the recruitment site is
transcriptionally competent, the result is
activation (CRISPRa) or repression/interfer-
ence (CRISPRi) of transcription. Although
this strategy has been applied in human cell
culture and animal models ( 4 , 5 ), the ulti-
mate task of employing CRISPRa to thera-
peutically rescue pathologic gene expression
has not been fully realized. Matharu et al.
use CRISPRa to restore the expression of
two haploinsufficient genes, single-minded 1
(Sim 1 ) and melanocortin 4 receptor (Mc 4 r),
to physiological amounts in mouse models
of severe early-onset obesity. Haploinsuffi-
ciency of either gene causes severe obesity
in humans, and previous work in mice es-tablished that SIM 1 and MC 4 R control eat-
ing behavior through their expression in the
hypothalamus ( 6 – 8 ); therefore, a relevant
therapeutic intervention would target gene
expression specifically in the hypothalamus.
Because Sim1 and Mc 4 r are expressed in
multiple tissues, an important first step was
to address whether it is feasible to modu-
late expression in a tissue-specific manner.
The authors tested two approaches, focus-
ing initially on Sim1: (i) Target CRISPRa to
the promoter of the remaining functional
Sim1 gene to enhance expression wher-
ever Sim1 was already active, and (ii) tar-
get CRISPRa to a 270 - kb distal enhancer
that controls Sim1 expression specifically
in the hypothalamus (see the figure). Both
approaches were employed in transgenic
animals expressing the CRISPRa reagents
(dCas9 fused to the transcriptional activa-
tor VP64), as well as recombinant adeno-
associated virus (rAAV)–mediated delivery
of CRISPRa directly into the hypothalamus.
In all cases, hypothalamic Sim1 expression
was restored to wild-type levels and the
mice did not become obese, demonstrat-
ing robust prevention of a haploinsufficient
phenotype by enhancing endogenous gene
expression. Interestingly, the authors found
that they could manipulate Sim1 expression
exclusively in the hypothalamus by target-
ing the hypothalamic enhancer instead of
the Sim1 promoter, indicating that to obtain
tissue-specific transcriptional modification,
CRISPRa will likely need to be deployed to
tissue-specific regulatory elements. Injec-
tion of rAAV-based CRISPRa into the hy-
pothalamus of Mc 4 r haploinsufficient mice
similarly prevented obesity, further demon-
strating the strength of this approach.
This strategy illustrates what could
emerge as an important new approach to
treating gene expression disorders and
raises the possibility of expanding the scope
of CRISPRa and CRISPRi technology to
treat diseases that involve pathogenic over-
expression of a gene, particularly in cancer.
For example, somatic mutations in a subset
of pediatric T cell acute lymphoblastic leu-
kemia (T-ALL) result in the formation of a
highly active enhancer that drives onco-
genic TAL 1 gene overexpression ( 9 ). More-Department of Human Genetics, University of Chicago,
Chicago, IL, USA. Email: [email protected]1 8 JANUARY 2 019 • VOL 363 ISSUE 6424 231
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