Tumor

Granzyme B

TRM

TEM

Figure 1. Effector Functions of Tissue-Resident Memory T Cells in the Breast. In triple-negativebreast cancers, the tissue resident memory T cells have higher effector functions for killing tumor cells than Tcells that enter the tissues.

tissue (as the authors demonstrated in afew cases of prophylactic mastectomiesfrom healthy women). Nevertheless, inthe age of immunotherapy, even rare anti-tumor immune populations may prove tobe clinically important in deciding whichpatients to treat. Specifically targetinggenes and pathways in TRM cells in TNBCpatients may improve responses in immu-notherapy that,despite intensive investiga-tion, have been underwhelming in clinicaltrials.

AcknowledgmentsWe thank the American Cancer Society (129098-

RSG-16-092-01-TBG), Simons Family Foundation,

822 Trends in Molecular Medicine, October 2018, Vol. 24,

and Chen-Zuckerberg Initiative (HCA-A-1704-

01668) for grants to N.N.

1Department of Genetics, The University of Texas MD

Anderson Cancer Center, Houston, TX, USA2Graduate School of Biomedical Sciences, The University

of Texas MD Anderson Cancer Center, Houston, TX, USA3Department of Melanoma Medical Oncology, The

University of Texas MD Anderson Cancer Center,

Houston, TX, USA4Department of Bioinformatics and Computational

Biology, The University of Texas MD Anderson Cancer

Center, Houston, TX, USA

*Correspondence: nnavin@mdanderson.org (N. Navin).

https://doi.org/10.1016/j.molmed.2018.07.006

References1. Savas, P. et al. (2018) Single-cell profiling of breast cancer T

cells reveals a tissue-resident memory subset associatedwith improved prognosis. Nat. Med. 24, 986–993

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2. Papalexi, E. and Satija, R. (2018) Single-cell RNA sequenc-ing to explore immune cell heterogeneity. Nat. Rev.Immunol. 18, 35–45

3. Neu, K.E., Tang, Q., Wilson, P.C. and Khan, A.A. (2017)Single-cell genomics: approaches and utility in immunology.Trends Immunol. 38, 140–149

4. Tirosh, I. et al. (2016) Dissecting the multicellular ecosystemof metastatic melanoma by single-cell RNA-seq. Science352, 189–196

5. Mackay, L.K. et al. (2012) Long-lived epithelial immunity bytissue-resident memory T (TRM) cells in the absence ofpersisting local antigen presentation. Proc. Nat. Acad.Sci. U. S. A. 109, 7037–7042

6. Wang, Z.-Q. et al. (2016) CD103 and intratumoral immuneresponse in breast cancer. Clin. Cancer Res. 22, 6290–6297

7. Jiang, X. et al. (2012) Skin infection generates non-migratorymemory CD8 TRM cells providing global skin immunity.Nature 483, 227–231

8. Ali, H.R. et al. (2014) Association between CD8+ T-cellinfiltration and breast cancer survival in 12,439 patients.Ann. Oncol. 25, 1536–1543

SpotlightEmerging Strategies forGenome Editing in theBrainDana V. Foss1,2,4 andRoss C. Wilson1,2,3,*

Despite the unparalleled therapeu-tic promise of genome editing, itscurative power is currently limitedby the substantial difficulty in deliv-ering DNA-cutting enzymes to thecells in need of correction. A recentstudy demonstrates the potentialfor the delivery of pre-assembledgenome-editing enzymes in theform of ribonucleoprotein com-plexes, which were used to rescuea mouse model of fragile Xsyndrome (FXS).

Genome editing has rapidly transformedbiomedical research and has demon-strated therapeutic promise via suc-cesses in tissue culture, ex vivo,embryonic editing, and animal modelsof human disease. For successful

translation, a genome-editing therapeuticmust be safe, effective, and ideallystraightforward to manufacture. DNAencoding the RNA and protein compo-nents of a CRISPR-derived genome edit-ing enzyme such as Cas9 can bedelivered by adeno-associated virus(AAV) with high efficacy, but safety maybe a concern and the manufacturing bur-den is substantial [1]. One emerging alter-native is the delivery of genome-editingenzymes in the form of a pre-assembledribonucleoprotein (RNA and protein, orRNP) complex. This approach is appeal-ing because it ensures a tight therapeuticwindow: the RNP will be degraded in lessthan 24 h. By contrast, viral expressioncan result in prolonged expression ofthe genome-editing enzyme that persistsfor days. This has been associated withan increased prevalence of unintendedoff-target edits compared with RNP-based editing [2]. The nuclear localizationsignal (NLS) has routinely been used toensure transport of RNP from the cytosolto the nucleus, but transporting a largegenome-editing enzyme from the cellexterior to the cytosol presents a distinctchallenge. Several strategies have beensuccessful in promoting the cellularimport of Cas9 RNP, such asmodificationof the Cas9 protein to include incidentallymembrane-disrupting NLS sequences[3], or appending negatively chargeddomains to the Cas9 protein to promoteits interaction with polymers that promotecellular entry [4]. The Murthy lab hasdeveloped an approach that uses anucleating gold nanoparticle conjugatedto single-stranded DNA to recruit Cas9RNP, all of which is coated in a cationicpolymer that facilitates delivery across thecell membrane, dubbed CRISPR-Gold[5].

The brain is an appealing site for initialforays into therapeutic genome editingbecause it is anatomically insular, allow-ing straightforward surgical access andproviding an immunoprivileged status

that ameliorates the risks associated withintroduction of viral vectors [1] and/orgenome-editing enzymes [6]. In a recentstudy by Lee and colleagues [7], the Leeand Murthy labs collaborated in usingCRISPR-Gold to deliver either Cas9 orthe analogous Cas12a (Cpf1) to themouse brain. CRISPR-Gold carryingeither Cas9 or Cas12a was stereotacti-cally injected into the mouse hippocam-pus or striatum, performing efficientgenome editing as detected by fluores-cent reporters. A mouse model of FXS,based on an Fmr1 knockout (KO), wasused for experiments probing the abilityof genome editing to treat autism. FXS isa common, inherited single-gene form ofautism spectrum disorder (ASD), anddrug treatments are largely inadequate.Importantly, the mGluR5 gene hasemerged as a promising candidate forgenetic therapy, since it can contributeto FXS as well as other ASDs. To testwhether reduction of mGluR5 coulddiminish autism-associated phenotypesin the FXS model mice, CRISPR-Goldbearing a Cas9 RNP targeting mGluR5was injected into the striatum. In treatedstriatal cells, 15% of mGluR5 loci weredisrupted, leading to a �40% reductionin mGluR5 mRNA or protein abundance,via qPCR or immunostaining, respec-tively. Behavioral studies of edited FXSmodel mice showed a marked reductionin two established hallmarks of mice withautistic phenotypes: marble-burying andspontaneous jumping. This promisingresult was bolstered by the observationthat CRISPR-Gold treatment had nodiscernible impact on mouse locomo-tion. Other tests for toxicity showedthat CRISPR-Gold treatment was notassociated with cell death in vivo orchanges in the properties of culturedneurons.

It is illustrative to evaluate these CRISPR-Gold results in comparison with AAV-mediated delivery, which was quicklyadopted by pioneering genome editors

Trends in

for use in the brain (Figure 1). Delivery ofAAV encoding Cas9 and its single guide(sg)RNA (Cas9/AAV) has been particu-larly successful in generating models ofneurodegenerative diseases and otherdiseases of the brain and nervous sys-tem. A 2016 report from the Zhang lab-oratory reported Cas9/AAV-mediatedediting of the MCP2 gene in the brainsof mice, resulting in disruptive editsin a majority (68%) of the cells in theinjected tissue. The observed robust viraldistribution throughout the tissue andediting in postmitotic neurons allowedgeneration of a mouse model of Rett’ssyndrome bearing the correspondingbehavioral phenotype [8]. AAV has alsobeen applied in therapeutic models; forexample, the Davidson laboratory editedthe disease-causing allele in a transgenicmouse model of Huntington’s disease,observing reductions in the levels ofmutant huntingtin protein of up to 80%following an injection of Cas9/AAV intothe brain [9]. A similar approach wasrecently reported for in vivo editing ofthe mutant alleles of the APP gene thatunderlies Alzheimer’s disease, anothercondition with dominant inheritance[10]. Cas9/AAV vectors were injectedinto the hippocampus of transgenic adultmice expressing multiple copies of thehuman mutant APP allele, and selectivelygenerated indels (1.3%) in the mutantallele allowing a decrease in pathogenicamyloid-b protein levels in the brains ofthe mice [10]. Neither example of Cas9/AAV editing disease-causing mutantalleles demonstrated an associated ther-apeutic phenotype in mice, as wasconvincingly demonstrated with theCRISPR-Gold phenotype in the recentreport by Lee and colleagues. However,the model systems differ, and it is rea-sonable to anticipate that Cas9/AAV-mediated editing might perform compa-rable editing in an FXS model system.

One apparent advantage of AAV-mediated delivery is that the viral particles

Molecular Medicine, October 2018, Vol. 24, No. 10 823

CRISPR-Gold

Strengths

Efficient edi�ng once in cellsStraigh�orward produc�on

Low off-target edi�ng

Limita�ons

Toxicity of co-delivered goldMay be immunogenic

Limited distribu�on

Viral vector

Strengths

Efficient cell entry & edi�ngRobust �ssue distribu�on Well-established

Limita�ons

May induce off-target editsCan be immunogenicManufacturing costsSize limits of cargo

Cas9

Delivery:

Figure 1. Delivery of Genome Editing Enzyme Cas9 to the Brain by CRISPR-Gold or Adeno-Associated Virus (AAV). Cas9 can be delivered to the brain in the form of an intact ribonucleoproteincomplex (magenta) via CRISPR-Gold (left), or as DNA instructions (green) encapsulated within a vector, suchas AAV. CRISPR-Gold comprises a gold nanoparticle (yellow) decorated with single-stranded DNA (blue) thatrecruits Cas9, all coated in a cationic polymer (black) capable of mediating cellular entry. By contrast, viraldelivery relies on the capsid proteins (gray) for access to the cell interior, which is followed by transcription andtranslation of the packaged DNA to produce the RNA and protein components constituting Cas9.

spread throughout the brain in mice andprimates. By contrast, RNP as deliveredin isolation [3] or by CRISPR-Gold[7] tends to edit only cells within an

824 Trends in Molecular Medicine, October 2018, Vol. 24,

area of several cubic millimeters. Thissuggests a potential hurdle for translation.Another potential concern related to theuse of CRISPR-Gold in humans is its

No. 10

introduction of heavy metal, which isknown to be toxic. However, this issueis tempered by the knowledge that thegold constitutes aminiscule fraction of thenanoparticle assembly by weight, andthat genome editing is ideally a one-timetreatment that avoids the accumulation ofgold that would be associated with atreatment that is repeatedly dosed. Withadditional development, RNP deliverymay prove itself as a leading strategyfor therapeutic genome editing of thebrain.

1Innovative Genomics Institute, University of California,

Berkeley, California 94720, United States2California Institute for Quantitative Biosciences,

University of California, Berkeley, California 94720, United

States3ORCID https://orcid.org/0000-0002-0644-55404ORCID https://orcid.org/0000-0003-2937-4703

*Correspondence: rosswilson@berkeley.edu (R.C. Wilson).

https://doi.org/10.1016/j.molmed.2018.07.008

References1. Colella, P. et al. (2018) Emerging issues in AAV-mediated in

vivo gene therapy. Mol. Ther. Methods Clin. Dev. 8,87–104

2. Kim, S. et al. (2014) Highly efficient RNA-guidedgenome editing in human cells via delivery of purifiedCas9 ribonucleoproteins. Genome Res. 24, 1012–1019

3. Staahl, B.T. et al. (2017) Efficient genome editing in themouse brain by local delivery of engineered Cas9ribonucleoprotein complexes. Nat. Biotechnol. 35,431–434

4. Wang, M. et al. (2016) Efficient delivery of genome-editingproteins using bioreducible lipid nanoparticles. Proc. Natl.Acad. Sci. U. S. A. 113, 2868–2873

5. Lee, K. et al. (2017) Nanoparticle delivery of Cas9ribonucleoprotein and donor DNA in vivo induceshomology-directed DNA repair. Nat. Biomed. Eng. 1,889–901

6. Leong, Chew Wei (2017) Immunity to CRISPR Cas9 andCas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol.Med. 10, e1408

7. Lee, B. et al. (2018) Nanoparticle delivery of CRISPR intothe brain rescues a mouse model of fragile X syndromefrom exaggerated repetitive behaviours.Nat. Biomed. Eng.2, 497–507

8. Swiech, L. et al. (2015) In vivo interrogation of gene func-tion in the mammalian brain using CRISPR-Cas9. Nat.Biotechnol. 33, 102–106

9. Monteys, A.M. et al. (2017) CRISPR/Cas9 editing of themutant huntingtin allele in vitro and in vivo. Mol. Ther. J.Am. Soc. Gene Ther. 25, 12–23

10. György, B. et al. (2018) CRISPR/Cas9mediated disruptionof the Swedish APP allele as a therapeutic approach forearly-onset Alzheimer’s disease. Mol. Ther. Nucleic Acids11, 429–440