Science - USA (2019-01-18)

INSIGHTS | PERSPECTIVES

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over, MYC gene expression in human B cell
acute myeloid leukemia (AML) was recently
shown to be dependent on a 1.7-megabase
distal enhancer element ( 10 ). Both stud-
ies demonstrated that disrupting these en-
hancer elements negatively affected cancer
cell survival, providing a precedent for de-
veloping CRISPRi as a therapeutic approach
to inactivate cancer-promoting enhanc-
ers. Although transcription factors such as
TAL1 and MYC are among the most potent
oncoproteins, targeting them with small-
molecule inhibitors has proven challenging.
The results presented by Matharu et al. sug-
gest that it should be possible to circumvent
protein-targeted therapies by quelling onco-
gene expression at its source—transcription.

A key advancement in the study by
Matharu et al. is their use of rAAV to deliver
CRISPRa reagents in vivo. For a CRISPR-
based therapeutic to be relevant for use in
humans, it will likely need to be packaged
within a virus and administered intrave-
nously, because most targeted cell types will
not be available for ex vivo manipulation and
implantation. rAAV is nonpathogenic and
displays a high delivery potential, making
it a viable option for effectively introducing
CRISPR reagents to human cells. CRISPRa
and CRISPRi approaches have the added
benefit of modulating gene expression with-
out modifying the genome, thereby avoiding
potential off-target mutations. Thus, pairing
CRISPRa with rAAV to treat a gene expres-
sion disorder in vivo is an important step
forward in the development of expression-
based therapeutics.
Although Matharu et al. demonstrate
that CRISPR-based up-regulation of a hap-
loinsufficient gene can prevent obesity, this
study also raises the important question
of whether a disease phenotype can be re-
versed. Because the authors administered
CRISPRa reagents to mice at 4 weeks of
age—before the onset of obesity—they did
not address the potential to rescue the

phenotype later in life. Many haploinsuffi-

cient disorders in humans are likely to be

therapeutically actionable only after the

disease phenotypes are partially or fully es-

tablished. Future experiments should test

the therapeutic benefit of targeting gene

expression with the goal of reversing a hap-

loinsufficient phenotype. Additionally, it is

important to recognize that many enhanc-

ers are dynamic, meaning that they may act

at specific developmental stages and change

their tissue specificity with time ( 11 ). Fortu-

nately, the authors were able to capitalize

on a developmentally stable tissue-specific

enhancer, although it is unclear how often

this will be the case for targeting enhancers

of other haploinsufficient genes.

Naturally occurring and pathogenic gene

regulatory DNA elements provide a tailored

therapeutic route to targeting gene expres-

sion. The results presented by Matharu et

al. underscore the importance of identifying

and carefully characterizing the enhancers

that control gene expression. Large-scale ef-

forts have identified thousands of putative

enhancers in hundreds of human cell types.

However, cell types representing diverse

disease states, particularly from human pa-

tients, remain understudied. Knowing the

full repertoire of gene regulatory elements

and their target genes ( 12 ) in these cell types

is likely to provide critical insight that can be

exploited for CRISPR-based therapeutic ap-

proaches to modify gene expression. j

REFERENCES AND NOTES

1. N. Huang et al., PLOS Genet. 6 , e1001154 (2010).


2. N. Matharu et al., Science 363 , eaau0629 (2019).


3. C.-H. Lau, Y. Suh, Transgenic Res. 27 , 489 (2018).


4. M. L. Maeder et al., Nat. Methods 10 , 977 (2013).


5. H. Zhou et al., Nat. Neurosci. 21 , 440 (2018).


6. J. L. Michaud et al., Hum. Mol. Genet. 10 , 1465 (2001).


7. M. J. Krashes et al., Nat. Neurosci. 19 , 206 (2016).


8. C. Vaisse et al., J. Clin. Invest. 106 , 253 (2000).


9. M. R. Mansour et al., Science 346 , 1373 (2014).


10. C. Bahr et al., Nature 553 , 515 (2018).


11. A. S. Nord et al., Cell 155 , 1521 (2013).


12. L. E. Montefiori et al., eLife 7 , e35788 (2018).

10.1126/science.aaw0635

IMMUNOLOGY

B cells, CMV,

and stem cell

transplant

Host antibodies help prevent

CMV dissemination after

bone marrow transplantation

By Maria-Luisa Alegre

H

ematopoietic stem cell transplanta-

tion (HSCT) is a potentially curative

therapy for various malignant and

nonmalignant conditions but can

be complicated by infections such

as reactivation of cytomegalovirus

(CMV). CMV is a ubiquitous DNA herpes

virus comprising various distinct strains

( 1 ). Around 60 to 90% of healthy adults are

seropositive, indicating past exposure to the

virus, although infections in healthy people

are often mild or asymptomatic. After ini-

tial acute infection, CMV enters a latent

phase, similar to other herpes viruses. It

has been suggested that CMV infection may

confer an immunological benefit, perhaps

explaining its prevalence, because com-

parison of seropositive and seronegative

individuals has shown that CMV infection

results in greater responses to the flu vac-

cine ( 2 ). The immune system is essential to

control the initial infection and to prevent

later CMV reactivation, as demonstrated by

the high incidence of CMV reactivation in

immunosuppressed patients such as HSCT

recipients. Whereas the incidence and se-

verity of CMV transcriptional reactivation

and cell-to-cell dissemination after HSCT

has substantially diminished since the

adoption of prophylactic or preemptive an-

tiviral therapies ( 3 ), CMV remains the most

important viral infection after HSCT, espe-

cially in high-risk patients (such as seropos-

itive recipients of seronegative donors), and

can lead to life-threatening CMV disease in

~10% of HSCT recipients ( 4 ). On page 288

of this issue, Martins et al. ( 5 ) make the

unexpected observation, in mice undergo-

ing bone marrow transplantation (BMT) as

a model of HSCT, that strain-specific CMV

antibodies made by host B cells play a cru-

cial role in preventing CMV dissemination

Section of Rheumatology, Department of Medicine,

University of Chicago, Chicago, IL 60637, USA.

Email: [email protected]

Haploinsufcient gene CRISPRa enhances expression

VP64

dCas9

Allele 1

Coding

exons

Tissue-speci1c enhancer

Nonsense mutation

mRNA

Allele 2

Allele 1

Allele 2

232 18 JANUARY 2019 • VOL 363 ISSUE 6424

Enhancing endogenous gene expression with CRISPRa
Most genes are biallelically expressed; however, heterozygous mutations can cause haploinsufficiency,
resulting in 50% less functional protein. Recruitment of CRISPRa to the endogenous promoter or enhancer of
the gene in mouse models causes up-regulation of the wild-type copy, leading to normal expression levels.

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