Cas9 system: A promising technology for the

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May 15, 2018 - The multiplex gene editing using the CRISPR technology in len- tivirus-transduced CAR .... improved specificity.65,66 Alternatively, different SpCas9 variants were evaluated for their .... DNA repair mechanism (highlighted in red) is observed to .... Bjurstrom CF, Mojadidi M, Phillips J, et al. Reactivating fetal ...
Received: 15 May 2018

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Revised: 15 May 2018

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Accepted: 15 May 2018

DOI: 10.1002/acg2.10

INVITED REVIEW

CRISPR/Cas9 system: A promising technology for the treatment of inherited and neoplastic hematological diseases Justin S. Antony1,2,3

s Lamsfus-Calle2 | Alberto Daniel| A.K.M. Ashiqul Haque1 | Andre

Moreno2 | Markus Mezger2 | Michael S.D. Kormann1 1

Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of T€ ubingen, T€ ubingen, Germany 2

University Children’s Hospital, Department of Paediatrics I, Hematology and Oncology, University of T€ ubingen, T€ ubingen, Germany

3

Department of Hematology, Oncology, Clinical Immunology, University of €bingen, Tu €bingen, Germany Tu Correspondence Michael S.D. Kormann, Translational Genomics and Gene Therapy in Paediatrics, University Children’s Hospital-Section I| Paediatric Infectiology & Immunology, €bingen, Wilhelmstrasse 27, University of Tu 72074 T€ ubingen, Germany. Email: [email protected] Funding information J€ urgen Manchot Stiftung; Europe Research Council Starting Grant, Grant/Award €ne Grant, Tu €bingen, Number: 637752; Fortu Grant/Award Number: 2412-0-0, 2485-0-0

Abstract The ongoing advent of genome editing with programmable nucleases, including zincfinger nuclease (ZFN), TAL effector nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9), have spurred the hematopoietic stem cell gene therapy (HSCGT). In particular, CRISPR/Cas9-mediated gene editing revealed promising outcomes in several preclinical disease models including inherited and neoplastic hematological diseases. In this review, we focused on the utilization of the CRISPR/Cas9 system as a possible treatment option for hemoglobinopathies and hematological tumors. We summarize the recent advances with CRISPR/Cas9 and its therapeutic potential for genome editing in cells from hematopoietic origin. We also critically discussed the limitations inherent to the CRISPR/Cas9 and possible alternatives for the improvement of genome editing. KEYWORDS

b-thalassemia, CRISPR/Cas9, gene editing, hematological tumors, sickle-cell disease

1 | INTRODUCTION

patients with primary immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies.1-3 However, the treatment caused

Recent technological advancements bring more attention and hope

the transactivation of proto-oncogene, and higher frequency of

to the field of cell and gene therapy. The delivery of therapeutic

random integration loads (5-20 million/kg body weight), which

genes or correction of mutated genes in target cells might provide

raise a possible safety concern related to insertional mutagene-

a possible cure for patients with inherited monogenic diseases and

sis.4,5 Moreover, the random integration of viral vectors result in

neoplastic tumors. In particular, the hematopoietic stem cell gene

either low or very high transgene expression often without expres-

therapy (HSC-GT) exhibited clinical benefit for several diseases, as

sion control of the cellular system.1-3 Therefore, the ideal HSC-GT

these gene-corrected stem cells were stably differentiated into

must overcome these limitations by ensuring targeted integration

multiple hematopoietic lineages. Deep knowledge on hematopoi-

of therapeutic transgene at safe-harbor, preferentially at the

etic system and well-established HSC transplantation protocols

endogenous locus to provide a transcription control by natural reg-

have profited the ex vivo gene therapy using autologous HSCs.

ulatory elements of the promoter. As HSC-GT is very personalized

Interestingly, the gene therapy clinical trials with viral-vector–

and need individualized manufacturing, the safety and precise gene

transduced autologous HSCs showed safety and efficacy for

editing is mandatory. The advancement on genome editing with programmable nucleases, including zinc-finger nuclease (ZFN), TAL effector

Markus Mezger and Michael S.D. Kormann: Shared Senior Authors.

Adv Cell Gene Ther. 2018;e10. https://doi.org/10.1002/acg2.10

wileyonlinelibrary.com/journal/acg2

© 2018 John Wiley & Sons Ltd

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nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9), have revolutionized gene therapy per

ET AL.

2 | CRISPR/CAS9 IN INHERITED NONMALIGNANT HEMATOLOGICAL BLOOD DISORDERS

se, and primarily HSC-GT.6 Several clinical and preclinical studies have investigated these gene editing tools in HSC-GT, and suc-

b-Hemoglobinopathies are congenital hematologic diseases, of which

cessfully demonstrated precise and targeted-transgene integration

approximately 56 000 newborns with b-thalassemia and 270 000

(TTI).6,7 Though ZFNs/TALENs-mediated gene therapy reached

newborns with sickle-cell disease (SCD) are affected worldwide.9 In

the clinical trial phase I/II before CRISPR/Cas9, the gene editing

general, b-hemoglobinopathies are monogenic diseases in which a

with the CRISPR/Cas9 system is under focus as it holds several

single gene is mutated.10 To date, over 300 mutations have been

8

described in the human b-globin gene (HBB).11 Since the year 1985,

Therefore, here we review the recent progress with CRISPR/

HBB gene locus was primarily targeted for gene editing because this

Cas9-mediated gene editing in the context of inherited and neo-

gene is associated with common genetic diseases including b-thalas-

plastic hematological diseases. We also critically discussed the

semia and SCD, and it holds the possibility of ex vivo gene correc-

limitations inherent to the CRISPR/Cas9 such as off-target

tion in HSCs due to its short gene length.12 From then, several gene

effects and poor efficiency of homology-directed repair. In addi-

editing tools including ZFNs and CRISPR/Cas9 have been tried to

tion, we have attempted to provide a possible alternatives for

make a clinically meaningful gene correction at HBB locus.13,14 Here,

the improvement on these limitations from the literature and per-

we have provided the studies that had utilized the CRISPR/Cas9

sonal views.

technology to correct b-hemoglobinopathies in chronological order

advantages including superior gene targeting and easy design.

(Table 1). There are 2 different approaches of gene modification for b-hemoglobinopathies: (1) gene correction where specific mutation is

T A B L E 1 Most relevant b-hemoglobinopathies studies carried out by utilizing CRISPR/Cas9 genome editing in human cells Type of modification Gene correction

Gene disruption

Cell line

Gene

Cas9 delivery

Repair template

Efficiency of gene editing

Reference

iPSCs

HBB (c.20 A>T)

pDNA

NA

75% NHEJ

15

iPSCs

HBB (IVS-1)

pDNA

PiggyBac

23% HDRa

30

iPSCs

HBB (IVS-2)

pDNA

dsDNA

17% HDRa

29

a

27

iPSCs

HBB (IVS-II 654 C>T)

pDNA

PiggyBac

12% HDR

iPSCs

HBB (c.20 A>T)

pDNA

dsDNA

40% HDRa

19

3PN hu. embryos

HBB (CD41/42 - 4 bp del)

mRNA

ssODNs

14% HDR

26

HSCs

HBB (Exon1 & c.20 A>T)

mRNA/RNP

AAV6

10% HDRa

16

a

iPSCs

HBB (CD41/42 - 4 bp del)

pDNA

ssODNs

5% HDR

iPSCs

HBB (CD41/42 - 4 bp del)

pDNA

dsDNA

57% HDR

HSCs

HBB (c.20 A>T)

mRNA

IDLV

20% HDR

22 25 18 a

17

HSCs

HBB (c.20 A>T)

RNP

ssODNs

33% HDR

iPSCs

HBB (CD41/42 - 4 bp del)

pDNA

dsDNA

NA

23

2PN hu. embryos

HBB (CD41/42 - 4 bp del)

RNP

ssODNs

25% HDR

24

HSCs

HBB (c.20 A>T)

mRNA/RNP

ssODNs

9% HDR

20

HSCs

HBB (CD41/42 - 4 bp del)

pDNA

ssODNs

54% HDR

21

iPSCs

HBB (Exon1 and 30 UTR)

pDNA

HBB cDNA

NA

28

HSCs

BCL11A (GATA 1 motif)

Lentivirus

-

NA

36

HSCs

HBG1/2

Lentivirus

-

77% NHEJ

41

K562

KLF1 (Exon 2, exon 3)

pDNA

-

23% NHEJ

39

HSCs

HBD-HBB

pDNA

-

31% NHEJ

42

HSCs

BCL11A (Exon 2)

pDNA/mRNA

-

13% NHEJ

35

MEL/ch11

LCR (HS2, HS3)

Lentivirus (LV)

-

NA

38

HSCs

HBA MCS-R2 enhancer

dsDNA

-

60% NHEJ

44

HSCs

HBG-HBD, HBD-HBB

pDNA

-

20% NHEJ

43

a Homology-directed repair (HDR) rate calculated by clone selection. HSCs, hematopoietic stem cells; iPSCs, induced pluripotent stem cells; 2PN hu. embryos, 2 pronuclear human embryos; 3PN hu. embryos, 3 pronuclear human embryos; ssODNs, single-stranded oligodeoxynucleotides; NHEJ, nonhomologous end joining; IDLV, integrase-deficient lentivirus; RNA, ribonucleoprotein; AAV, adeno-associated virus.

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corrected with a repair template or adding a HBB transgene to the

of the HPFH by deleting the d- and b-globin genes (13 kb), or delet-

endogenous locus, and (2) gene disruption in which the expression

ing the c-globin promoter (13 bp) was tested and observed to exhi-

of fetal-type hemoglobin (HbF) is induced by repressing genes that

bit higher HbF expression.41-43 The targeted deletion of 3.5 kb in c-

are involved in HbF repression.

d intergeneic region and the 7.2 kb in Corfu region, which induces

The gene correction strategy for b-hemoglobinopathies using the

HPFH was observed to be a potential therapeutic target for HbF

CRISPR system was tested in different human cell sources including

reactivation.43 Alternatively, the CRISPR/Cas9-mediated induction of

patient/donor-derived induced pluripotent stem cells (iPSCs), HSCs

a-thalassemia by disrupting the MCS-R2 a-globin enhancer was

and human embryos (Table 1). For this approach, studies were

observed to rectify the imbalance of globin chains that typically

mostly targeting the sites near the SCD mutation (HBB:c.20A>T,

observed in b-thalassemia.44 For the easy understanding, different

p.E6V; rs334)

15-20

and most common b-thalassemia mutation, that

is, 4-bp deletion at codon 41/42 (HBB:c.124_127delTTCT/HBB:

genes that targeted by disruption strategy and their outcomes were illustrated in Figure 1.

c.126_129delCTTT) which is prevalent in East Asia and Southeast Asia.21-26 Moreover, a study had tried to correct a common b-thalassemia splicing variant IVS2-654 (HBB:c.316-197C>T).27 In addition to the mutation-specific correction, various studies had explored the

3 | CRISPR/CAS9 IN NEOPLASTIC HEMATOLOGICAL DISEASES

possibility of universal gene correction by adding a HBB transgene at the endogenous locus by TTI strategy.16,28-30 Studies have used dif-

In normal physiological condition, hematopoiesis is a tightly regulated

ferent Cas9 delivery systems such as pDNA, mRNA, and RNP, and

process in which HSCs are proliferate and differentiate into mature

surprisingly the efficiency of gene correction varies dramatically even

blood cells. However, dysregulation in certain regulatory pathways

for the same delivery system irrespective of the cell line tested and

leads to hematological cancers including leukemia, lymphoma, and

donor template used. For example, pDNA-encoded Cas9 exhibited

myeloma.45 The clinical trials with chimeric antigen receptor (CAR)-

different gene correction rate ranging from 5% to 57% (Table 1).

modified T cells have created big hope for treating some of the

However, systematic comparison of these 3 delivery system showed

blood cancers with gene therapy.46 CARs are synthetic immune

that mRNA- and RNP-mediated delivery is effective to attain high

receptors and one of the best products of gene-fusion technique,

31

level of gene targeting.

which are comprised of an extracellular single-chain variable frag-

Human erythropoiesis undergoes 2 “globin switches” during the

ment (ScFv) to recognize tumor antigen and an intracellular chimeric

development: (1) from embryonic to fetal globin (HbF) in uterus, and

signaling domain for T-cell activation.47 Gene editing technologies in

(2) from fetal to adult globin (HbA) immediately after birth. The pro-

the context of CAR T-cell therapy were primary employed for uni-

cess is strictly controlled by molecular regulators including the locus

versalization of allogeneic CAR T cells and targeted CAR-gene inte-

control region (LCR) and different repressors/enhancers.32 Earlier

gration at desired locus. Though most of the CAR T clinical trials

review has documented that the presence of HbF reduces the dis-

have used the autologous T cells, the low quality and insufficient T

ease severity for b-hemoglobinopathies.33 Thus, researchers have

cells derived from blood cancer patients results in unsuccessful treat-

focused on reactivating HbF as a possible treatment for b-hemoglo-

ment, and increases the price of the therapy due to the custom

binopathies by gene disruption of the transcription factors and key

modification. In this background, researchers have utilized different

regulators involved in the HbF to HbA switching process. For the

gene editing tools to overcome these hurdles by generating universal

gene disruption strategy, the resurgence of HbF is stimulated by

allogeneic CAR T cells (off-the-shelf), that a single CAR T-cell pro-

long term repression of specific genes (knock-out), which involved in

duct could be delivered to many patients.48

HbF silencing. The current reactivation therapy is focused on

The real excitement of gene editing to treat blood cancers was

BCL11A, a direct transcriptional repressor of HbF, as it is a potential

started when 2 children with acute lymphoblastic leukemia (ALL)

therapeutic target for b-hemoglobinopathies.34 Importantly, the gene

exhibited the therapeutic profile. In this study, investigators have

disruption strategy is more favored by the nonhomologous end-join-

used TALENs to disrupt the expression of CD52 and T-cell receptor

ing (NHEJ) repair pathway that leads to high level of desired gene

(TCR) in the T cells of healthy donor and generated universal CAR T

editing. The CRISPR/Cas9 mediated knock-down of BCL11A was

cells which were then transplanted to non–HLA-matched ALL

observed to increase the HbF levels in HSCs.35,36 Interestingly, the

patients (off-the-shelf therapy/CAR sharing).48 In similar with

genetic disruption of BCL11A erythroid enhancer is an approach

TALENs, the CRISPR/Cas9 technology is being used to possibly treat

from CRISPR Therapeutics (CTX001) which is expected to reach

some blood cancers by molecular targeting in T cells. In addition,

clinic trial soon in Europe.37

several preclinical studies were performed with murine and human

Other possible targets, such as KLF1, ZBTB7A/LRF, and LCR

blood cancer cell lines to identify the potential molecular targets for

genes, were directed by the CRISPR system. The repression of KLF1

several blood cancer subtypes (comprehensively reviewed in.49 Sev-

induced the HbF expression, whereas other targets were either not

eral studies have used the CRISPR/Cas9 system to target specific

altered or tested.32,38,39 Furthermore, as the coinheritance of heredi-

gene(s) which are linked to blood cancer and modulated their expres-

tary persistence of fetal hemoglobin (HPFH) with b-thalassemia alle-

sion through knock-out or knock-in in order to explore the therapeu-

viates its clinical manifestations,40 the CRISPR-mediated recreation

tic potential of the same.

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F I G U R E 1 Network of molecular regulators taking part in the fetal (HbF) to adult (HbA) hemoglobin switch, and novel CRISPR/Cas9 genome disruption approaches to increase HbF as a therapy for b-hemoglobinopathies: b- globin gene cluster in chromosome 11 constituted by the locus control region (LCR) and an assembly of genes e, cG, cA, d, and b. a-Globin gene cluster in chromosome 16 comprising the regulatory elements (RE) and the genes f, a1, and a2. MyB, KLF1, BCL11A, SOX6, GATA1, and ZBTB7A/LRF represent the genes regulating cglobin expression. CRISPR/Cas9 gene disruptions of (1) the c-globin repressor BCL11A, (2) KLF1, and (3) the binding site of c-globin repressors would increase HbF levels. (4) Recreation of the hereditary persistence of fetal hemoglobin (HPFH) by deleting the d- and b-globin genes (13 kb), or deleting the c-globin promoter (13 bp)

The multiplex gene editing using the CRISPR technology in len-

endogenous gene expression, oncogenesis, and lack of control over

tivirus-transduced CAR T cells have generated universal CAR T cells

CAR expression. To overcome these hurdles, a recent study have

by long-term repression of endogenous TCR (TRCA) and HLA class I

used the CRISPR/Cas9 system and targeted TRAC locus of T cells,

(B2M) molecules, and increased the T-cell activity by a stable disrup-

inserted CD19-CAR construct, and replaced the endogenous T-cell

50

tion of programmed cell death 1 (PD-1).

An independent study tar-

receptor.7 The study have demonstrated that CRISPR-based CAR T

geting the same 3 genes (TRCA, B2M, and PD-1) reported similar

cells were more effective in removing cancer cells than CAR T cells

results and highlighted that the CAR T cells with double knock-out

that generated with viral vectors. Moreover, the viral vector medi-

(TRCA, B2M) resulted in 100% survival rate in a tested xenograft

ated overexpression of CAR results in a constitutive T-cells activa-

51

A subsequent study have generated allogeneic uni-

tion and thereby T-cell exhaustion by expression of some inhibitory

versal CAR T by quadruple gene disruption where TRCA, B2M, PD-1,

receptors including PD-1, whereas the expression of CRISPR-based

and cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) genes

CAR T cells were physiologically controlled by the same genetic reg-

were targeted, and CTLA-4 knock-out using the one-shot CRISPR

ulators that control the endogenous TCR expression.7

mouse model.

system exhibited the high relevance for clinical gene editing.52

In addition to the T-cell–directed immunotherapy against blood

Besides to the multiplex strategy, other studies have individually tar-

cancers, researchers have also edited HSCs using the CRISPR/Cas9

geted genes using the CRISPR system to enhance the outcome of

technology. Unlike B-cell lymphoma with CD19 antigen, acute mye-

CAR T-cell therapy.53,54

loid leukemia (AML) cells lack of specific surface antigen which cre-

The transfer of CARs in cancer patient’s T cells are typically

ates difficulties when targeting with CAR T-cell immunotherapy.

achieved through the transduction of c-retroviral and lentiviral vec-

However, Kime et al. demonstrated that CRISPR-based CD33 knock-

tors which randomly integrated into several parts of the genome. As

out in HSCs enabled the CD33-directed CAR T cells against AML

discussed earlier, random integrations are not the preferred events

cells with increased efficacy and reduced toxicity by not disrupting

in an ideal gene therapy due to the possibility of disrupting the

the normal myeloid function.55 Along with the therapeutic targeting,

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the CRISPR technology was employed to create disease models such

RNP showed undetected levels of off-targets for the selected

as creation of AML mouse model with cancer-driving mutations in

sgRNA.37 These data clearly signify the importance of the transient

multiple target genes,56,57 in vitro model for natural killer-cell lym-

expression system to reduce the off-targets. Compared to the RNP

phoma (NKCL) with the knock-out of important tumor suppressor

system, the Cas9 mRNA offer further optimization that can be possi-

genes.58 Interestingly, the simplicity of the CRISPR technology

bly exploited to reduce off-target such as “kinetics of expression.”

enabled the researchers to uncover the genes responsible for resis-

Though mRNA is known for transient expression, several factors

tance of anticancer drug imatinib in chronic myeloid leukemia by

influence their expression kinetics including target cell types and

59

Overall,

their cellular machinery, chemical modifications, and GC content in

the combination of CAR T-cell therapy with the CRISPR technology

the mRNA transcript. For clinically meaningful gene editing, the opti-

opens the prospect to treat different blood cancers by improved

mal Cas9 mRNA should result in higher expression for very short

immunotherapy with favorable features including generation of uni-

time as this expression nature would directly correlate with reduced

versal CAR T cells, improved and endogenously controlled T-cell

off-target effect (kinetics 1 [K1]). However, the longer expression of

activity.

Cas9 mRNA might result in higher fold of off-targets (kinetics 2

high-throughput screening with 121 413 sgRNAs library.

[K2]). We propose to utilize this nature of Cas9 mRNA to reduce the off-targets. A complete summary of this part of the review is given

4 | C H A L L E N G E S O F C R I S P R /C A S 9 I N

in Figure 2A.

CLINICAL USE Although CRISPR-Cas9 have enormous potential for clinical use, this

4.2 | Homology-directed repair efficiency

technology still retains a few major challenges associated with safety

With CRISPR/Cas9, we have 3 possible editing approaches (1) gene

and efficiency. In this part of the review, we critically discussed

disruption (2) gene correction, and (3) gene addition. The desirable

these limitations in the context of HSCs gene editing with possible

editing strategy depend on the purpose and nature of the target dis-

options for the improvement.

ease. In HSCs, the gene disruption is relatively feasible due to the dominance of NHEJ repair pathway. However, gene correction and gene addition with donor templates depend on homology-directed

4.1 | Off-Target Effect

repair (HDR), which is inefficient and appears to occur at very low

The therapeutic gene editing of HSCs and T cells with CRISPR/Cas9

frequency. To circumvent this limitation, several studies were

must ensure increased genome-wide specificity with reduced off-tar-

attempted to increase the HDR efficiency by using HDR enhancers,

gets as the system associated with nonintended genetic alter-

NHEJ inhibitors, cell-cycle regulators, and optimally designed donor

ations.15,60 Ideally, CRISPR/Cas9 would result in a targeted genome

templates. Here, we itemized these molecules based on their nature

editing without any detectable genome-wide off-targets. This is a

(genetic/small chemical molecule/donor design) with their relative

very important criteria for editing the HSCs since the edited cells

reproducibility, as inconsistent results were observed among differ-

must be able to proliferate and differentiate normally. Several opti-

ent studies using the same molecules (Figure 2B).

mizations were reported to reduce the off-targets either at the level

The overexpression of genes involved in the HDR DNA repair

of sgRNA design or at the level of different Cas9 variants through

pathway, including RAD51, RAD52, and dn53BP1, were reported to

site-directed mutagenesis.

increase the knock-in efficiency.71,72 Among these, the role of

At the level of sgRNA, the genome-wide specificity was improved

RAD51 was found to play a prominent role as it is a key regulator

by the design of truncated-sgRNA with the length of 17 or 18 nucleo-

for the HDR pathway, while its expression was observed to be stim-

tides instead of 20 bp.61,62 Similarly, the design of sgRNA with optimal

ulated by RS-1, a HDR enhancer.73 Alternatively, the molecules that

63

A new study found

involved in NHEJ were inhibited either chemically or genetically to

that the sgRNA with DNA-RNA chimera reduce the off-targets.64

GC content increased the on-target efficiency.

improve the frequency of HDR. Primarily, DNA ligase IV, a key

Recently, developed methods including Guide-Seq and Circle-Seq

enzyme of NHEJ was targeted with a small molecule SCR7 that has

were utilized to preselect the single-guide RNAs (sgRNAs) with

inhibited the NHEJ and improved the HDR.74,75 In a subsequent

65,66

Alternatively, different SpCas9 variants were

study, authors have targeted DNA ligase IV by SCR7, gene silencing,

evaluated for their improved specificity such as high-fidelity Cas9

and overexpression of genes (E1B55K and E4orf6) which involved in

improved specificity.

62

nickase

proteasomal degradation of DNA ligase IV, and noted the enhanced

Cas9 (D10A mutation), dCas9 with FokI endonuclease (Cas9 with

HDR level.76 Though earlier reports showed promising HDR results

(quadruple substitutions, N497A/R661A/Q695A/Q926A), 67

D10A and H840A substitution),

enhanced specificity Cas9 (triple

substitutions, K848A/K1003A/R1060A),

68

and hyper-accurate Cas9

(quadruple substitutions, N692A/M694A/Q695A/H698A).

69

for SCR7 treatment, later investigations showed a controversial outcome and also reported the toxicity of this compound.73,77The suppression of other key molecules in the NHEJ pathway were also

Interestingly, transiently expressed Cas9 mRNA resulted in

studied to increase HDR-mediated genome editing, such as siRNA-

reduced off-targets compared to long-term expression with the len-

mediated suppression of KU70/80, use of CYREN gene to inhibit

tiviral system.70 Recent preclinical study in CD34+ HSCs using Cas9

NHEJ through KU70/80, inhibition of 53BP1 with ubiquitin-binding

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(A)

F I G U R E 2 (A) Cas9 mRNA kinetics and off-target effects in CD34+ HSCs: Graphical representation of 2 different Cas9 mRNA kinetics (K1 and K2) in HSCs and their impact on off-target effects. The longest duration of Cas9 mRNA (K2) will result in higher off-target cleavage. The optimization of different chemical modifications and other parameters might result in optimal kinetics (K1) that reduce the off-target effects. (B) DNA repair pathway mechanism and molecules involved: To attain higher targeted transgene integration at targeted endogenous locus several chemical compounds and genetic molecules were tested. The overexpression of HDR pathway molecules (highlighted in green) and inhibition of molecules involved NHEJDNA repair mechanism (highlighted in red) is observed to increase the HDR efficiency

(B)

proteins (i53), and use of small-molecule inhibitors (NU7441 and KU-0060648) of DNA-PK to reduce NHEJ.76,78-80 All studies were

5 | CONCLUSION AND FUTURE DIRECTIONS

showed substantial improvement in HDR rate. However, studies with small-molecule inhibitors showed high level of variance in dif-

CRISPR/Cas9 has great potential for precise gene therapy. The

ferent cell lines.81 These data clearly demand the substantial

promising preclinical results from CRISPR Therapeutics AG, a gene

improvement on compound screening and further validation in target

editing company, for the treatment of b-thalassemia and SCD has

cells such as HSCs.

given us great hope. However, the technology still needs operational

The HDR pathway is enriched during G2/M phase of the cell

improvements in order to make the safe step into the clinic which

cycle. Therefore, studies have utilized the cell-cycle regulators

includes confronting the preexisting immunity against Cas9 protein

such as cyclin (CCND1; G1/S transition) and nocodazole (G2/M

in human,87 ex vivo selection of gene-edited cells, and measurement

synchronization) to increase HDR efficiency. The nocodazole-

of CRISPR/Cas9-associated genotoxicity in highly proliferating

mediated cell-cycle synchronization and delivery of Cas9 RNP has

CD34+ HSCs.

82

increased HDR up to 38%.

Moreover, new improvements in the

donor template design as a viral vector or as a ssODNs were observed to increase transgene integration or gene correction. A

ETHICS STATEMENT AND CLINICAL IMPLICATIONS

latest study showed that homology-mediated end joining (HMEJ)

Despite of the great potential of CRISPR/Cas9, several important

has enhanced the HDR efficiency where the AAV repair template

ethical concerns were raised on its possible abuse with germline

was optimally designed with longer homology arms and sgRNA-

editing including “designer babies”. Moreover, recent reports of gene

binding site.83 A detailed protocol to edit HSCs for HDR using

editing in nonviable human embryo raised serious discussion on ethi-

84

In addition,

cal implications and demands stringent regulations from authorities

the strategic design and chemical modifications in oligo repair

(24, 26). Aside from the ethical concerns, CRISPR/Cas9 still need

templates have raised the HDR in vitro.85,86 All these studies

further improvements on their safety level to be used in human

were attempted to increase the HDR rate; however, there is still

germ lines. However, the use CRISPR/Cas9 to correct genetic

room for improvement.

defects by carefully editing HSCs and other somatic cells possess

CRISPR/cas9 and AAV was published very recently.

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greater clinical impact. A recent review by Kohn et al., thoroughly assessed the clinical implementation of CRISPR/Cas9 gene editing with insights from ethics and regulatory aspects.88

ACKNOWLEDGMENTS We thank Mr. Brain Weidensee and Ms. Merve for the help with the proof-reading of the draft.

AUTHOR CONTRIBUTIONS J.S.A. wrote the complete manuscript. A.H., A.L.C., and A.D.M., have contributed with the figures and tables, and also proof-read the draft. M.M., and M.S.D.K. drafted the final version of the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTERESTS None of the authors have any competing interests in the manuscript.

FUNDING SOURCES €ne grant (to J.S.A., No. J.S.A. was financially supported by fortu 2485-0-0) and Europe Research Council Starting Grant-ERC StG (to M.S.D.K., No. 637752), and. A.H., was supported by ERC StG € rgen Man(to M.S.D.K., No. 637752). A.L.C. was supported by Ju € ne grant (to M.M., No. chot-Stiftung. A.D.M., supported by a fortu 2412-0-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ORCID Justin S. Antony

http://orcid.org/0000-0003-1241-2013

Michael S.D. Kormann

http://orcid.org/0000-0003-3492-0393

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How to cite this article: Antony JS, Haque AKMA, LamsfusCalle A, Daniel-Moreno A, Mezger M, Kormann MSD. CRISPR/Cas9 system: A promising technology for the treatment of inherited and neoplastic hematological diseases. Adv Cell Gene Ther. 2018;e10. https://doi.org/10.1002/ acg2.10