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
