cells

Review
CRISPR-Based Gene Therapies: From Preclinical to
Clinical Treatments
Marine Laurent 1,† , Marine Geoffroy 2,† , Giulia Pavani 3, * and Simon Guiraud 2, *

1 INTEGRARE, UMR_S951, Genethon, Inserm, Univ Evry, Université Paris-Saclay, 91190 Evry, France
2 SQY Therapeutics, 78180 Montigny-le-Bretonneux, France
3 Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
* Correspondence: pavanig@[Link] (G.P.); [Link]@[Link] (S.G.);
Tel.: +1-2674261222 (G.P.); +44-(0)1865-285880 (S.G.)
† These authors contributed equally to this work as co-first authors.

Abstract: In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and
CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited
disorders affecting different organ systems, such as blood and muscles. Both hematological and neu-
romuscular genetic disorders benefit from genome editing approaches but face different challenges
in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem
cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In
the last decade, many clinical trials were initiated and are now delivering encouraging results. The
recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and
transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights
the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR
therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular
field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and
animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed
up preclinical development of therapeutic solutions. Several corrective interventions have been
proposed to genetically restore dystrophin production using the CRISPR toolbox and have demon-
strated promising results in different DMD animal models. Although these advances represent a
significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are
still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector
Citation: Laurent, M.; Geoffroy, M.; doses, together with safety and immunological concerns. Collectively, the results obtained in the
Pavani, G.; Guiraud, S. CRISPR-Based hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for
Gene Therapies: From Preclinical to patients affected by these debilitating conditions. As each field suffers from different and specific
Clinical Treatments. Cells 2024, 13, 800. challenges, the clinical translation of CRISPR therapies may progress differentially depending on
[Link]
the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of
Academic Editor: Toshifumi Yokota these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions.
This review provides insights into the diverse applications of CRISPR-based technologies in both
Received: 26 March 2024
preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare
Revised: 3 May 2024
advances in both fields while highlighting current trends, difficulties, and challenges to overcome.
Accepted: 5 May 2024
Published: 8 May 2024
Keywords: CRISPR/Cas9; gene editing; blood disorders; neuromuscular disorders; β-hemoglob-
inopathies; Duchenne muscular dystrophy

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and
Site-specific endonucleases can modify the genome at a specific DNA locus, represent-
conditions of the Creative Commons
ing a promising tool to cure genetic disorders, including blood and muscular diseases [1].
Attribution (CC BY) license (https://
Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)
[Link]/licenses/by/
belong to the first generation of gene editing tools, comprising a customizable DNA-binding
4.0/).

Cells 2024, 13, 800. [Link] [Link]

 Cells 2024, 13, x FOR PEER REVIEW 2 of 26

Cells 2024, 13, 800 2 of 27

[1]. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases
(TALENs) belong to the first generation of gene editing tools, comprising a customizable
DNA-binding
domain fused todomain
a DNA fused to amodule
cleavage DNA cleavage
[2]. Frommodule [2].to
the 1990s From the 1990s
the early 2010s, tomany
the early
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contributed contributed to
to the identification of the identification
a microbial of aimmune
adaptive microbialsystem
adaptive immune
against phage
system against
infection phage
that relied oninfection that relied
short repeated on short(clustered
sequences repeated sequences (clustered regu-
regularly interspaced short
larly interspaced
palindromic short
repeats, palindromic
CRISPR) and repeats, CRISPR) and
an RNA-guided DNA annuclease
RNA-guided DNA nuclease
(CRISPR-associated
(CRISPR-associated
protein 9, Cas9) [3–7]protein
(Figure9,1).
Cas9)
The[3–7] (Figure 1).of
development The development
genome editingofmethods
genome based
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methods basedtechnologies
CRISPR/Cas9 on CRISPR/Cas9 technologies
dramatically dramaticallythe
revolutionized revolutionized
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ther-
apy field, rapidly expanding its therapeutic
expanding its therapeutic applications [8–10]. applications [8–10].

Figure 1. Timeline highlighting the important milestones in CRISPR gene editing field (black ar-
Figure 1. Timeline highlighting the important milestones in CRISPR gene editing field (black arrows).
rows). Achievements in CRISPR-based blood disease research are specified in blue and advents in
Achievements in CRISPR-based
CRISPR-mediated blood disease
Duchenne muscular research
dystrophy are specified
research in blue and advents in CRISPR-
are in green.
mediated Duchenne muscular dystrophy research are in green.
The CRISPR/Cas9 system uses a chimeric single-guide RNA (sgRNA) to direct an
The CRISPR/Cas9 system uses a chimeric single-guide RNA (sgRNA) to direct an
endonuclease (Cas9) to specific DNA targets, where it generates double-strand breaks
endonuclease (Cas9)
(DSBs) [9] (Figure to These
2A). specific DNA
DSBs cantargets, wherebyitthe
be repaired generates double-strand
cell through breaks
two competing
(DSBs) [9] (Figure 2A). These DSBs can be repaired by the cell through
pathways: the error-prone non-homologous end joining, which generates insertions andtwo competing path-
ways: the error-prone non-homologous end joining, which generates insertions
deletions at the cutting site, or the more precise homology-directed repair, which requires and dele-
tions
a DNAat thetemplate
cutting site, or the more
to introduce precise
specific homology-directed
modifications repair, which
[11]. Pioneering requires a DNA
CRISPR/Cas9 ap-
template to introduce specific modifications [11]. Pioneering
proaches have been successfully used in several clinical trials for lung CRISPR/Cas9 approaches
cancer
have been successfully
(NCT02793856) [12] used
and infor
several clinical trials for lung
β-hemoglobinopathies cancer (NCT02793856)
(NCT03655678, [12] and
NCT03745287,
for β-hemoglobinopathies (NCT03655678, NCT03745287, NCT04443907),
NCT04443907), resulting in the first FDA-approved CRISPR therapy ([Link]/news- resulting in the
first FDA-approved CRISPR therapy ([Link]/news-events/press-announcements/
events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-
fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease,
disease, accessed on 8 December 2023). Despite these successes, it is becoming accessed on 8 Decem-
more evi-
ber 2023). Despite these successes, it is becoming more evident that Cas9-induced
dent that Cas9-induced DSBs can lead to genotoxic effects that include large insertions/de- DSBs can
lead to genotoxic
letions, effectstranslocation,
chromosomal that include large insertions/deletions,
and chromothripsis [13–16],chromosomal translocation,
raising long-term safety
and chromothripsis [13–16], raising long-term safety concerns.
concerns.
Base editing provides a more precise and efficient method for making single-base
changes compared with traditional gene editing approaches [17] (Figure 2B). Potentially,
around 60% of pathogenic point mutations could be corrected by base editors (Bes). Bes
were developed by fusing a Cas9 nickase (nCas9) to a deaminase [18,19]. Unlike tradi-
tional CRISPR/Cas9, Bes do not generate DSBs; instead, the deaminase domain promotes
base conversions at specific genomic locations upon sgRNA targeting. Cytosine Bes (CBE)
can convert a C:G to a T:A base pair, while adenine Bes (ABEs) can convert A:T to G:C.
CellsCells
2024,2024, 13, x FOR PEER REVIEW
13, 800 4 of 26
3 of 27

Figure2. [Link]
Figure Theexpanding
expandingCRISPR/Cas9
CRISPR/Cas9 toolbox.
toolbox. (A)
(A) CRISPR/Cas9,
CRISPR/Cas9,a agene-editing
gene-editingtechnology,
technology,is is
composedofoftwo
composed twoessential
essentialcomponents:
components: aa guide
guide RNA
RNA (sgRNA)
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targetaadesired
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DNAlocus, and
locus, and
the endonuclease Cas9 (CRISPR-associated protein 9). CRISPR/Cas9 leads to a double-stranded
the endonuclease Cas9 (CRISPR-associated protein 9). CRISPR/Cas9 leads to a double-stranded DNA
DNA break, allowing genomic modifications. (B) Base editing: nCas9 are fused to cytidine or aden-
break, allowing genomic modifications. (B) Base editing: nCas9 are fused to cytidine or adenosine
osine deaminases (cytosine base editor or adenine base editor) to induce substitutions or base edits
deaminases (cytosinedouble-strand
without inducing base editor orbreaks.
adenine base
(C) editor)
Prime to induce
editing: nCas9substitutions
can be fusedortobase edits without
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inducing
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(RT) and extended prime(C)editing
Primeguide
editing:
RNAnCas9 can be
(pegRNA) fused
(prime to a reverse
editor) transcriptase
to mediate targeted
small
(RT) and insertions,
extendeddeletions, and base-to-base
prime editing guide RNA conversions.
(pegRNA)Twin prime
(prime editing
editor) is an innovative
to mediate targeteditera-
small
tion of prime
insertions, editor and
deletions, protein based on two
base-to-base pegRNAs Twin
conversions. for programmable
prime editingdeletion, substitution,
is an innovative inser-of
iteration
tion, editor
prime or inversion
proteinofbased
largeronDNA
two sequences.
pegRNAs for (D)programmable
dCas9 can be fused to transcriptional
deletion, activatorsor
substitution, insertion,
(CRISPRa) or (E) transcriptional repressors (CRISPRi) to precisely modulate gene expression. (F)
inversion of larger DNA sequences. (D) dCas9 can be fused to transcriptional activators (CRISPRa)
dCas9 can be fused to epigenetic effector domains as histone/DNA modifier to alter the epigenetic
orstate
(E) transcriptional
at precise DNArepressors (CRISPRi) toCas9;
locations. dCas9—dead precisely modulateCas9.
nCas9—nickase gene expression. (F) dCas9 can be
fused to epigenetic effector domains as histone/DNA modifier to alter the epigenetic state at precise
DNA locations. dCas9—dead
2. CRISPR-Based Cas9; nCas9—nickase
Gene Editing Cas9.
for Blood Disease
The only curative treatment for many inherited blood disorders is allogeneic trans-
Base editing provides a more precise and efficient method for making single-base
plantation of healthy hematopoietic stem/progenitor cells (HSPC). This procedure is lim-
changes compared with traditional gene editing approaches [17] (Figure 2B). Potentially,
ited by the availability of compatible donors and is associated with serious complications
around 60% of pathogenic point mutations could be corrected by base editors (Bes). Bes
and life-threatening risks [39,40]. Gene therapy strategies based on autologous transplan-
were developed by fusing a Cas9 nickase (nCas9) to a deaminase [18,19]. Unlike traditional
tation of ex vivo transduced HSPCs have been successfully developed. Despite the clinical
CRISPR/Cas9, Bes do not generate DSBs; instead, the deaminase domain promotes base
benefits of these therapies, retro/lentiviral gene addition can result in subtherapeutic or
conversions at specific genomic locations upon sgRNA targeting. Cytosine Bes (CBE) can
unregulated transgene expression and carries an intrinsic risk of insertional mutagenesis.
convert a C:G to a T:A base pair, while adenine Bes (ABEs) can convert A:T to G:C. Even
Hence, the ability to correct patient HSPCs using genome editing technologies represents
if anew Bes with less sequence constraints [20], C-to-G conversion capabilities (glycosy-
game changer for the field. HSPCs are routinely mobilized and isolated from patients
lase base editors,
and healthy GBEsThey
donors. [21,22]),
can and different ex
be modified editing
vivo windows have methodologies
with different been developedand[23],
many pathological mutations cannot be targeted with this technology, including small
and large deletions and multi-nucleotide changes. New editing tools have been gener-
Cells 2024, 13, 800 4 of 27

ated to overcome these limitations, including prime editing [24], CRISPR/integrases [25],
CRISPR/transposases [26], and large serine recombinases [27].
Prime editors (PE) can introduce specific mutations without inducing DSBs and
perform insertions up to 44 bp [24] (Figure 2C). This system is composed of a nCas9 fused
with a reverse transcriptase, and a prime editing gRNA (peg-RNA) that serves both as a
guideRNA and a template to install the desired modification at a specific site. By using a
pair of partially complementary pegRNAs, it is possible to increase insert size up to 110 bp
or generate large genomic deletions (paired-peg, TWIN-PE) [28], though larger cassettes
cannot be delivered using this system alone.
Serine site-specific recombinases like Bxb1 can integrate, invert, and excise large DNA
fragments [29] by promoting the recombination of attP and attB sites. This reaction is
irreversible and unidirectional, making this system ideal for stably integrating expression
cassettes into the genome where one of the sites is present [30,31]. This observation
prompted the development of novel editing tools like PASTE (programmable addition via
site-specific targeting elements), which consists of a PE fused with Bxb1 [25]. PASTE can
insert DNA payloads up to 35 Kb at specific genomic locations and could be beneficial for
diseases involving large genes.
Apart from its capability to integrate, modify bases, or delete DNA sequences, the
CRISPR/Cas9 system can be leveraged to modify gene expression (Figure 2D–F). Dead
Cas9 (dCas9) can bind to specific DNA sequences by sgRNA targeting but has no nuclease
activity. dCas9 can be combined with various enzymes like transcriptional repressors
(CRISPR interference, CRISPRi) and activators (CRISPR activation, CRISPRa) to modulate
gene expression over several orders of magnitude [32]. After binding, CRISPRi creates a
steric hindrance that prevents transcription factors (TF) from attaching to the promoter
region of the targeted gene, resulting in decreased expression. The system is more effective
when transcriptional repressor domains (such as Krüppel-associated box, KRAB [33]) are
fused to dCas9. Inversely, the CRISPRa system utilizes transcriptional activator domains
(such as VP64, RTA, VP64, HSF1, and p65) to recruit RNA polymerase or TFs to promote
and increase the expression of downstream genes [34,35]. Gene transcription can be altered
at the epigenetic level by modulating DNA accessibility or chromatin structure at specific
sites. Different epigenetic enzymes have been employed in combination with dCas9 to
manipulate DNA methylation in gene promoters. For example, DNA methylases (DNMT)
or demethylases (TETF1) have been used to modify the methylation status of specific CpG
islands, therefore changing gene expression [36]. Chromatin accessibility can be altered
by recruiting fusion Cas9 proteins containing histone modifiers such as histone acetylases
(P300) that facilitate chromatin opening [37] or histone deacetylases (HDAC) that allow
histones to wrap DNA more tightly [38].
In this review, we will summarize main preclinical and clinical applications of CRISPR-
based technologies, focusing on monogenic blood disorders and muscular dystrophy,
comparing advances in both fields, and highlighting current trends, difficulties, and chal-
lenges to overcome.

2. CRISPR-Based Gene Editing for Blood Disease

The only curative treatment for many inherited blood disorders is allogeneic transplan-
tation of healthy hematopoietic stem/progenitor cells (HSPC). This procedure is limited
by the availability of compatible donors and is associated with serious complications and
life-threatening risks [39,40]. Gene therapy strategies based on autologous transplanta-
tion of ex vivo transduced HSPCs have been successfully developed. Despite the clinical
benefits of these therapies, retro/lentiviral gene addition can result in subtherapeutic or
unregulated transgene expression and carries an intrinsic risk of insertional mutagenesis.
Hence, the ability to correct patient HSPCs using genome editing technologies represents
a game changer for the field. HSPCs are routinely mobilized and isolated from patients
and healthy donors. They can be modified ex vivo with different methodologies and
reconstitute the entire blood system when transplanted [41]. These intrinsic properties
Cells 2024, 13, 800 5 of 27

of HSPCs, together with the extensive experience acquired through decades of lentiviral
gene therapies, have accelerated the development of gene editing approaches for many
hematological disorders. In this review, we will focus on two families of inherited blood
diseases: β-hemoglobinopathies and primary immunodeficiencies.

2.1. β-Hemoglobinopathies
β-hemoglobinopathies are conditions affecting the β chain of hemoglobin, the main oxygen
carrier in humans. In adults, the hemoglobin tetramer is composed of four chains—two α-
and two β-globin subunits (HbA, α2 β2 )—both necessary to transport oxygen from the
lungs to the tissues by the red blood cells (RBCs) [42]. β-hemoglobinopathies are the most
frequent monogenic disease, affecting 400,000 births worldwide each year, the main forms
being sickle cell disease (SCD) and β-thalassemia [43].
SCD is caused by an A-to-T mutation in the β-globin gene (HBB) resulting in the forma-
tion of hemoglobin S (HbS, β6Glu→Val ) [44]. Under low-oxygen tension, HbS polymerizes,
making erythrocytes more rigid and with a characteristic sickle shape. These sickled
RBCs trigger hemolytic anemia and vaso-occlusion events, causing ischemic damage to
tissues and leading to acute pain crisis and organ failure [45]. In contrast, β-thalassemia is
caused by a wide range of mutations, including large deletions that decrease the amount
of functional β-globin protein produced. The imbalance between β and α-chains causes
precipitation of α-globin in RBCs, resulting in hemolysis and impaired erythropoiesis [46].
Despite different pathophysiology, both disorders can be ameliorated by the coin-
heritance of benign mutations that increase levels of fetal hemoglobin (HbF, α2 γ2 ), as
γ-globin can replace β-globin function and has antisickling properties [43,47]. For these
reasons, many gene editing approaches for β-hemoglobinopathies rely on γ-globin reac-
tivation. To achieve this goal, nucleases can target transcriptional repressors of γ-globin
genes (HBG1/2) like B-cell lymphoma/leukemia 11A (BCL11a) or inhibitory regions in
HBG1/2 promoters. Many clinical trials were started in the past decade leveraging different
CRISPR/Cas9-based tools (Table 1). Recently, the FDA approved the first CRISPR/Cas9
based treatment for severe SCD and transfusion dependent β-thalassemia (TDT)—Exa-cel
(Casgevy) commercialized by Vertex Pharmaceuticals and CRISPR Therapeutics. This
editing treatment consists of autologous HSPCs electroporated ex vivo with Cas9/gRNA
mRNA to disrupt the erythroid-specific enhancer of BCL11a. Specifically, the BCL11A
locus harbors three DNase I hypersensitivity sites (DHS), serving as erythroid-specific
enhancers. These sites are situated at distances of +62, +58, and +55 kb from the transcrip-
tional start site [48]. Among these, the DHS at +58 kb is crucial for BCL11a expression in
erythroid cells [49,50]. Two clinical trials were initiated in 2018 for SCD (NCT05329649;
NCT03745287; NCT05951205) and TDT (NCT05356195; NCT03655678) (Table 1). Initial
findings reported successful engraftment of edited HSPCs (80% BCL11a alleles) one year
post-treatment. This resulted in significant production of HbF, with a reduction in blood
transfusions and vaso-occlusive events (in SCD patients) [51]. Two analogous strategies de-
vised by Bioray (BRL-101) and Edigene (ET-01) to disrupt the +58 DHS of BCL11a are under
clinical evaluation for TDT (BRL-101: NCT04211480; NCT05577312; NCT04205435; ET-01:
NCT04390971; NCT05752123; NCT04925206) (Table 1). During the 18-month follow-up for
BRL-101 (NCT04211480), two patients exhibited successful engraftment of modified HSPCs,
achieving an editing rate of 85% in the bone marrow. This led to a clinically meaningful
rise in HbF levels that translated to transfusion independence [52]. These results were also
achieved in ten TDT patients [53]. Preliminary results from ET-01 also showed promising
outcomes [54]. ZFNs were also utilized to disrupt the GATA-1 binding site in BCL11a locus
in TDT (NCT03432364) (Table 1). Despite an initial success, a decline in γ-globin synthesis
was observed in treated patients two years post-administration, requiring resumption of
blood transfusions [55,56].
An alternative approach to reactivate HbF is to disrupt the BCL11a binding site on the
γ-globin locus. In TDT patients, transplantation of HSPCs modified with CRISPR/Cas9 at
the γ-globin promoter (ChiCTR2100052858; ChiCTR2100053406) increased HbF production,
Cells 2024, 13, 800 6 of 27

eliminating the necessity for blood transfusions [57,58] (Table 1). Alternatively, Cas12
nuclease has been successfully utilized to disrupt repressors binding sites on HBG1/2 pro-
moters [59]. This editing strategy showed high editing efficiency, with a 40% increase in HbF
in vivo, and is currently under clinical investigation for TDT (NCT05444894, NCT06041620)
and SCD (NCT04853576) (Table 1). Because of their superior safety profile and multiplexing
properties, BEs have emerged as novel tools to reactivate γ-globin genes. This could be
achieved by disrupting repressors binding sites at the HBG1/2 promoters or by generating
de novo DNA motifs for transcriptional activators. Antoniou et al. used BEs to generate
several mutations associated with hereditary persistence of HbF and compared their impact
on γ-globin expression [60]. In this study, the generation of a binding site for KLF1 using
ABE showed the greatest HbF reactivation in SCD cells. Beam Therapeutic also used ABE
to generate a point mutation in HBG1/2 promoters to restore γ-globin expression. This
approach is currently being used in a clinical trial for SCD (NCT05456880) (Table 1).

Table 1. Clinical trials of blood diseases using CRISPR/Cas9 gene editing system.

Disease Clinical trial Therapeutic approach Editor Name Sponsor Study Phase
Gene disruption of BCL11A Sangamo Therapeutic
NCT03653247 ZFN BIVV003 Phase 1/2
using ZFN (Richmond, CA, United States)
NCT05329649 CTX001
NCT03745287 Gene disruption of BCL11A Vertex Pharmaceuticals Inc. Approved
using CRISPR-Cas9 CRISPR-Cas9 Exa-cel
NCT05951205 (Boston, MA, United States)
Gene disruption of BCL11A Novartis pharmaceuticals
NCT04443907 CRISPR-Cas9 OTQ923 Phase 1
using CRISPR-Cas9 (Basel, Switzerland)
SCD Gene disruption of γ-globin
Beam Therapeutics Inc.
NCT05456880 promoter using adenine base ABE BEAM-101 Phase 1/2
(Cambridge, MA, United States)
editor
NCT04853576 Gene disruption of γ-globin CRISPR- EDIT-301 Editas Medicine, Inc.
Cas12a Phase 1/2
promoter using CRISPR-Cas12a (Cambridge, MA, United States)
Gene correction using Graphite Bio
NCT04819841 CRISPR-Cas9 CEDAR Phase 1/2
CRISPR-Cas9 and AAV6 (San Francisco, CA, United States)
Gene correction using
NCT04774536 CRISPR-Cas9 CRISPR_SCD001 Mark Walters, MD Phase 1/2
CRISPR-Cas9 and ssODN
Gene disruption of γ-globin Sangamo Therapeutics
NCT03432364 ZFN ST-400 Phase 1/2
promoter using ZFN (Richmond, CA, United States)
NCT05356195 Gene disruption of BCL11A Vertex Pharmaceuticals Inc. Approved
NCT03655678 using CRISPR-Cas9 CRISPR-Cas9 CTX001 (Boston, MA, United States)
NCT04211480 Gene disruption of BCL11A Bioray Laboratories
NCT05577312 using CRISPR-Cas9 CRISPR-Cas9 BRL-101 Phase 1
(Boston, MA, United States)
NCT04390971
NCT05752123 Gene disruption of γ-globin EdiGene Inc.
promoter using CRISPR-Cas9 CRISPR-Cas9 ET-01 (Guangzhou, China) Phase 1
NCT04925206
ChiCTR2100052858 Gene disruption of γ-globin Guangzhou Reforgene Medicine
ChiCTR2100053406 promoter using CRISPR-Cas9 CRISPR-Cas9 RM-001 Phase 1
β- (Guangzhou, China)
thalassemia Gene disruption of γ-globin CRISPR- Editas Medicine, Inc.
NCT05444894 EDIT-301 Phase 1/2
promoter using CRISPR-Cas12a Cas12a (Cambridge, MA, United States)
Institute of Hematology & Blood
Gene disruption of γ-globin CRISPR-
NCT06041620 VGB-Ex01 Diseases Hospital Interventional
promoter using CRISPR-Cas12b Cas12b
(Tianjin, China)
Gene disruption of γ-globin
Glycosylase Bioray Laboratories
NCT05442346 promoter using glycosylase base - Phase 1/2
base editors (Shanghai, China)
editors
Gene correction of CVS-654 Bioray Laboratories Phase 1/2
NCT04205435 CRISPR-Cas9 -
mutation using CRISPR-Cas9 (Shanghai, China) Completed
Allife Medical Science and
NCT03728322 Gene correction in induced CRISPR-Cas9 - Technology Co., Ltd. Early phase 1
HSCs using CRISPR-Cas9
(Beijing, China)

Epigenetic activation of γ-globin has been proposed [35], both with zinc finger com-
bined with transcriptional activator VP64 [61,62] and dCas9 fused with acetylase (p300) [37];
however, their γ-globin reactivation efficiency is lower compared with genome editing
approaches.
Direct correction of the SCD mutation in HBB has been attempted, leveraging the
homology directed repair pathway. Different corrective templates have been used as DNA
donors in combination with CRISPR/Cas9, including adeno associated virus (AAV) [63]
or single-stranded oligodeoxynucleotides (ssODN) [64]. This approach was further de-
veloped [65] to initiate a clinical trial for SCD (NCT04819841) [66] (Table 1). However,
Graphite Bio halted the trial in early 2023 after observing complications in the first pa-
tient dosed, including prolonged low blood cell counts requiring transfusion and growth
factor support.
Cells 2024, 13, 800 7 of 27

Based on similar methods, gene replacement approaches that rely on the integration
of the β-globin gene into the α-globin locus have been devised [67,68]. These studies suc-
cessfully restored HbA expression in thalassemic and SCD erythroblasts. Such approaches
might benefit patients with large deletions spanning the γ- and β-globin locus where HbF
reactivation is not a viable therapeutic option.
BEs cannot directly correct the A>T mutation present in SCD. However, ABEs have
been used to modify the SCD allele to a non-pathogenic β-globin variant (β6Glu→Ala , Hb-
Makassar) [69,70]. An engineered ABE nuclease edited between 45% to 80% of the SCD
alleles in patient HSPCs, resulting in up to 72% of Hb-Makassar production in differentiated
erythroid cells [70]. The diverse mutational spectrum in β-thalassemia poses challenges to
the development of corrective therapies. Hardouin et al. proposed a base editing approach
to treat a common disease-causing point mutation in HBB (IVS1-110 [G>A]) that efficiently
rescued β-globin production [71]. Because of their precise editing activity, PEs can correct
the SCD mutation [24]. Everette et al. delivered an optimized ePegRNA and PE3max as
mRNA into SCD-HSPCs and achieved up to 41% of allele correction. Analysis of engrafted
cells isolated 4 months after transplantation showed expression of HbA in 42% of human
red cells [72]. Rather than relying on the ex vivo correction of HSPCs, Li et al. developed a
novel in vivo modification strategy to correct the underlying SCD mutation. After HSCP
mobilization, a helper-dependent adenoviral vector (HDAd) encoding the PE system was
administrated intravenously in an SCD mouse model (CD46/Townes). Viral transduction
corrected up to 40% of SCD alleles in vivo and increased HbA synthesis to similar levels [73].
While safety aspects of this delivery method need to be further explored, the direct modifi-
cation of HSPCs in vivo eliminates the requirement of myeloablation/transplantation, a
critical limitation for many gene editing therapies.
Overall, CRISPR/Cas9-derived tools have generated innovative and efficient treat-
ments for β-hemoglobinopathies. These studies are paving the way for a broader applica-
tion of this ground-breaking technology to many other genetic disorders.

2.2. Primary Immunodeficiencies

Primary immunodeficiencies are rare diseases comprising more than 130 genetic disor-
ders that affect the function and/or development of the immune system [74]. Some of these
diseases, such as adenosine deaminase-severe combined immunodeficiency (ADA-SCID)
and X linked-SCID (SCID-X1), have been successfully treated with viral gene therapy [75,76].
However, retroviral and lentiviral vectors carry the intrinsic risk of oncogenic insertional
mutagenesis, and therefore alternative strategies are needed. Moreover, many genes in-
volved in primary immunodeficiencies require a thoroughly regulated expression and
would benefit from a gene editing approach. Because pathological mutations are rare
and scattered along the gene body, most editing strategies for these disorders rely on the
targeted integration of a corrective cDNA at the endogenous locus. For many immunod-
eficiencies, corrected cells have a selective advantage over mutated ones. Even if gene
knock-in is not efficient in HSPCs, low correction levels could be sufficient to treat the
disorder in patients. In this section, we will describe genome editing approaches developed
for a subset of primary immunodeficiencies.
X-linked hyper-IgM syndrome (XHIM) is caused by mutations in the CD40 ligand
(CD40L) gene that result in ineffective antibody production, recurrent infections, and
autoimmunity in male infants. Ectopic expression of CD40L has been associated with
lymphoma in mice [77], therefore lentiviral approaches would not be recommended for
this disease. Proof-of-principle studies were performed using Cas9 and other nucleases
to integrate CD40L cDNA under the control of its endogenous promoter in primary T
cells [78,79] and in healthy HSPCs [80]. Importantly, CD40L activity was restored in
gene-edited cells in vitro and in vivo, without signs of aberrant differentiation. Recently,
large-scale good manufacturing practice (GMP)-compliant production processes have been
developed, bringing this therapy closer to clinical application [81].
Cells 2024, 13, 800 8 of 27

RAG1 is another gene involved in primary immunodeficiencies that requires tight

regulation. Recombination activating genes (RAGs) are involved in V(D)J recombination
and are required for correct lymphocyte development and function. A clinical trial using a
lentiviral approach is currently underway (NCT04797260); however, constitutive and/or
heterogenous expression of RAG1 raises safety concerns about potential immune dysreg-
ulation [82]. Using Cas9 and an AAV6 with a corrective cDNA, Castiello et al. restored
RAG1 expression and activity in patient HSPCs, leading to the generation of mature T and
B cells in transplanted mice [83].
Chronic granulomatous disease (CGD) is a group of genetic disorders in which neu-
trophils are unable to kill certain bacteria and fungi. The X-linked form of the disease
(X-CGD) is caused by mutations in the CYBB gene. Different editing strategies were pro-
posed to correct X-CGD in HSPCs, using nucleases with AAV6 vectors or ssODN as donor
templates [84–86]. These studies indicated that preserving the first intron of CYBB is crucial
to achieve a normal level of expression and restore effective ROS production [86]. Unfortu-
nately, gene correction rates need to be extensively optimized to achieve therapeutic effects
as corrected X-CGD cells do not have a selective advantage in vivo [87].
Most p47-CGD patients carry a specific deletion (∆GT) in exon 2 of the NCF1 gene,
making this form of CGD a good candidate for gene editing therapies. Two studies
have shown correction of the ∆GT mutation using nucleases in combination with a DNA
template [88,89], however efficiency must improve to reach therapeutic benefits in preclini-
cal models.
Severe congenital neutropenia (SCN) is a rare inherited disease that impairs neutrophil
maturation and is frequently caused by autosomal-dominant mutations in the ELANE gene.
Production of an abnormal protein results in neutropenia, despite the presence of one
intact ELANE allele. Induction of frameshift alleles with CRISPR/Cas9 can trigger non-
sense mediated decay and potentially relieve neutrophil maturation arrest [90,91]. An
allele-specific knockout strategy to precisely target the mutated copy of ELANE has been
proposed. By targeting heterozygous sites of single-nucleotide polymorphisms (SNPs)
associated with ELANE mutations, Sabo et al. were able to rescue neutrophil development
and function [92].
CD3δ-SCID is caused by biallelic mutations in the autosomal CD3D gene that result in
a profound deficiency of circulating mature T cells, often leading to infant mortality. CD3D
is essential for the productive assembly of TCR complexes, playing an important role in
T cell maturation [93]. In a recent study, McAuley et al. delivered ABE to CD3δ-SCID
HSPCs to correct the disease-causing CD3D c.202C>T mutation [94]. High editing efficiency
was achieved in these cells, together with functional correction and restoration of T cell
maturation. Edited cells persisted 16 weeks in xenografts and displayed normal T cell
receptor repertoires, making this approach a promising treatment for CD3δ-SCID.
Wiskott–Aldrich syndrome (WAS) is an X-linked disorder caused by mutations in
the WAS gene that accounts for approximately 3% of all primary immunodeficiencies [95].
Clinical manifestations depend on the underlying mutation, ranging from mild to se-
vere persistent thrombocytopenia, autoimmunity, increased susceptibility to infections,
or congenital neutropenia [96]. Lentiviral gene therapy has been successfully used for
WAS [97,98], but bleeding and autoimmune episodes were only partially resolved by the
treatment. Several knock-in strategies have been developed using different nucleases and
DNA donors in pluripotent stem cells and cell lines [99,100]. Recently, Rai and al. restored
WAS function in patient HSPCs by targeting WAS cDNA to its endogenous locus, using
Cas9/gRNA and AAV6 [101]. A side-to-side comparison was also performed between
genome edited and lentivirus transduced cells. Interestingly, transduced cells showed
improved functionality but to a much lesser extent compared with gene edited cells. Lim-
ited correction of B-cells, platelets, and myeloid cells was observed in the lentiviral group,
possibly due to suboptimal expression of WAS protein. In light of these promising results,
preclinical studies are on the way to further assess the safety and efficacy of this therapeutic
approach [102].
Cells 2024, 13, 800 9 of 27

3. CRISPR-Based Gene Editing for Duchenne Muscular Dystrophy

Similarly to blood diseases, the last decade witnessed the advent of CRISPR-base
gene editing in neuromuscular disorders, notably in the Duchenne muscular dystrophy
(DMD) field. From the rapid generation of helpful cellular and animal models to pre- and
clinical development of innovative therapeutic options, CRISPR gene editing technology
has revolutionized DMD research.
DMD is a fatal X-linked neuromuscular disorder characterized by severe and progres-
sive muscle weakness and wasting due to degeneration of skeletal, smooth, and cardiac
muscle [103]. Affecting more than 300.000 males worldwide, with a 1:5000 incidence in
male newborns [104], DMD is the most prevalent genetic muscular disorder in humans.
This orphan disease is caused by a variety of different mutations (mainly deletions (≈68%),
point mutations (≈20%), and duplications (≈11%)) in the DMD gene (OMIM 300377,
Xp21.2-p21.1) [105], known as the largest human gene (79 exons over 2.2 Mb of genomic
DNA) (Figure 3A). The DMD gene encodes for dystrophin (Uniprot P11532) [106], an
essential 427 kDa cytoplasmic protein critical for the maintenance of the biomechanical
properties of fiber strength, flexibility, and stability in skeletal muscle [107]. Acting as a
molecular shock absorber, the dystrophin protein establishes a mechanical link between the
extracellular matrix and the actin cytoskeleton through the dystrophin-associated protein
complex (DAPC) and allows myofibers to cope with repeated cycles of muscle contrac-
tion and relaxation. The absence of dystrophin destabilizes the DAPC and consequently
weakens the link between cytoskeleton and extracellular matrix, triggering muscle cell
dysfunction, muscle cell necrosis, and subsequent dystrophic tissue changes resulting
in fibro-fatty replacement of skeletal muscle. Affected boys typically develop the first
signs of motor dysfunction around the age of 2–3 years with abnormal gait, weakness
in proximal muscles and calf muscles, pseudo hypertrophy, and difficulties in running,
jumping, and climbing stairs. These symptoms progress relentlessly, loss of ambulation
occurs around the age of 10–12 years [108], followed by progressive generalized muscle
paralysis including respiratory insufficiency and cardiomyopathy [109]. Most patients
decease in young adulthood, with a median survival of 28.1 years (95% CI 25.1, 30.3) [110],
as a result of cardiac and respiratory failure. Despite exhaustive clinical attention for
respiratory support, management of cardiac complications, and corticosteroid treatment,
there is currently no cure for this devastating disease. The urgency to seek a cure for DMD
has resulted in parallel efforts to develop exon skipping [111,112], termination codon read
through [113] and dystrophin gene therapy [114], and non-dystrophin strategies [115].
Among all, anti-sense oligonucleotide (ASOs)-based exon skipping therapies aiming to
restore a functional reading frame by skipping one or more exons to produce a functional
dystrophin, are one of the most promising therapeutic options for DMD. Several exon-
skipping drugs were recently approved and, despite their success, new generation of ASOs
with improved chemistries, distribution, and efficiency such as the peptide-conjugated
phosphorodiamidate morpholino oligomer (P-PMO) [116] and Tricyclo-DNA (tcDNA) [117]
are currently required and under investigation in clinical trials for DMD. Acting at the
DNA level, CRISPR/Cas9 technology offers novel and promising opportunities in DMD
gene therapy.
 skipping drugs were recently approved and, despite their success, new generation of
ASOs with improved chemistries, distribution, and efficiency such as the peptide-conju-
gated phosphorodiamidate morpholino oligomer (P-PMO) [116] and Tricyclo-DNA
(tcDNA) [117] are currently required and under investigation in clinical trials for DMD.
Cells 2024, 13, 800 10 of 27
Acting at the DNA level, CRISPR/Cas9 technology offers novel and promising opportuni-
ties in DMD gene therapy.

Figure 3. Dystrophin structure and CRISPR/Cas9 strategies for DMD. (A) Structural organization of
the full-length wild-type dystrophin composed of 79 exons: the actin-binding N-terminal domain, the
hinge domains (H1–H4), a rod domain consisting of 24 spectrin domains (R1-R24), and a cysteine-rich
domain (CRD) next to a carboxy-terminal domain (CTD). The out-of-frame DMD ∆52 mutation (dele-
tion of exon 52) is one of the most common mutations in DMD patients. (B) CRISPR/Cas9 mediated
therapeutic strategies for DMD: NHEJ method (exon deletion, exon skipping, exon reframing), HDR
method (exon knock-in), base editing, prime editing, and CRISPR activator.
Cells 2024, 13, 800 11 of 27

3.1. CRISPR/Cas9 and Generation of DMD Study Models

To study the pathophysiology of the disease and to evaluate the efficacy of therapies
prior to clinical trials, cellular and animal models are mandatory. Only few immortalized
patient-derived muscle cell lines are currently available [118] as DMD muscle biopsies
are difficult to obtain. CRISPR/Cas9 technology allowed researchers to directly generate
human DMD lines with specific genetic mutations (Table 2). In the past years, several
novel immortalized DMD muscle cell lines were generated with the deletion of exons
50 [119], 51 [119], 52 [119,120], and 53 [119]. Recently, Tremblay’s lab produced DMD lines
with specific point mutations in exons 9, 20, 35, 43, 55, and 61 using prime editing [121].
These cell lines and associated subsequent assays [120,122,123] represent powerful tools to
evaluate and optimize therapeutic strategies, screen for effective drugs, and gain insights
in the disease pathophysiology.
Similarly, CRISPR/Cas9 offered a simple and efficient method to generate new animal
models of DMD (Table 2). The mdx mice is the most widely used DMD model and presents
a spontaneous point mutation in exon 23 of the Dmd gene, resulting in a premature stop
codon [124]. However, the mdx mouse develops a moderate and nonprogressive myopathy,
with a slightly shorter lifespan, and fails to recapitulate many symptoms observed in DMD
patients. Several murine models carrying specific exonic deletions were generated using
CRISPR/Cas9, such as ∆Ex52 [125], ∆Ex50 [126], ∆Ex45 [125], ∆Ex44 [127], ∆Ex43 [125],
or the large deletion ∆Ex8-34 [128]. Murine DMD models suffering from a non-sense
mutation in the Dmd exon 20 [129] and frameshift mutation [130] were also produced
by CRISPR and base editing. Importantly, all these strains phenocopied the mdx mice
and suffered from the same limitations. Moreover, these DMD models are of limited
use to test human sequence-dependent therapies such as exon skipping and gene editing
approaches. Consequently, humanized DMD mouse strains (hDMD mouse) such as the
hDMD∆52/mdx [131] and the hDMD∆45/mdx [132] were produced. Partially humanized
mice were similarly generated, replacing exon 45 and its adjacent introns with the human
sequences ((hEx45KI-mdx44) [133]. Although splicing mechanisms might be different, these
CRISPR-generated DMD models provide unvaluable in vivo platforms to assess human
sequence-dependent exon 44, 45, and 51 skipping drugs and gene editing approaches.
Rat models of DMD recapitulate more faithfully the human DMD pathophysiology,
allow behavioral experiments with high statistical power, and may represent a useful
alternative to mice. The first Dmdmdx rat was generated by TALEN [134] and suffered
from a severe muscle and cardiac pathology, reflecting some lesions and functional ab-
normalities observed in DMD patients. Using a two sgRNA pairs approach, the Relaix’s
lab successfully generated the R-DMDdel52 rat model, with a premature stop codon in
exon 52 of the rat Dmd gene [135]. Nakamura et al. also reported the generation of a
Dmd ∆Ex3-16 rat using SpCas9 and two sgRNA [136], and a Dmd ∆Ex45 rat model was
recently produced [137]. In the near future, humanized DMD rat models may certainly
serve preclinical drug development.
With an intermediate size and low maintenance costs, rabbit is another interesting
animal model of DMD. The first DMD rabbit was produced by co-injecting SpCas9 mRNA
and two gRNAs targeting the DMD exon 51 into rabbit zygotes [138]. This DMD ∆Ex51
rabbit model suffered from reduced dystrophin expression, histological defects, impaired
physical activity, cardiomyopathy, and premature death. At the crossroads between small
and large models, this novel rabbit DMD model could be valuable for preclinical studies.
In addition to rodent models, larger animal models of DMD such as pig, dog, and
monkey have also been produced using CRISPR systems. Porcine models have a number
of practical advantages and are phenotypically and phylogenetically closer to humans.
Several porcine models of DMD, such as the DMD ∆Ex52 pig [139], the DMD ∆Ex27
Chinese Diannan pig [140], or the DMD mEx13 [141], have been described and exhibited
a severe muscular dystrophy similar to human DMD but suffered from a faster disease
progression. DMD pigs are an attractive in vivo animal model, but their long gestational
period, costly maintenance, and reduced survival makes their preclinical use challenging.
Cells 2024, 13, 800 12 of 27

The golden retriever muscular dystrophy (GRMD) dog [142] and several other
breeds [143,144] suffered from spontaneous DMD genetic defects and closely mimicked
the human disease [145]. Oh et al. reported the generation of a DMD dog with a 57 bp
deletion in exon 6 by nuclear transfer using CRISPR/Cas9-edited somatic cells [146]. While
expensive and limited by the low number of phenotypically variable individuals, DMD
dogs represent a useful large animal model for preclinical studies.
Monkeys have mostly been used for safety evaluation and pharmacological studies
in the DMD field. Recently, the first DMD rhesus monkeys (Macaca mulatta) model with
various InDel mutations in exon 4 and 46 was generated [147]. These animals showed
dystrophin reduction and some histological defects. To date, this study is the only published
preliminary assessment of a DMD monkey model.
The ideal animal model should display a robust phenotype over generations, have the
same genetic defect, and mimic key hallmarks and progression of the human pathology.
None of the 60 animal models of DMD previously described fulfilled these requirements.
Each animal model presents key advantages and limitations and should be used for specific
applications. As an example, murine models are indicated for in vivo drug screening, while
dogs and monkeys are appropriate for assessing immune responses and toxicology. CRISPR
technologies have facilitated the generation of novel animal models of DMD, speeding up
preclinical development of therapeutic solutions and their translation to DMD patients.

Table 2. CRISPR-mediated animal models of DMD.

DMD
Model Muta- Target Nuclease Advantages Limitations Reference
tion
∆Ex50 I49 and I50 - Low cost [119]
human
immortalized - Easily maintainable - Cell type number limited
Cell

∆Ex51 I50 and I51 SpCas9 - Wide number of mutations - Does not recapitulate the human [119]
myoblasts cell
line ∆Ex52 I51 and I52 - Human genetic context disease [119,120]
∆Ex53 I52 and I53 - High high-througput screening [119]
Ex20 Ex20 BE3 [129]
Ex23 Ex23 CjCas9 - Low cost [130]
∆Ex43 I42 and I43 - Easily maintainable [125]
∆Ex44 I43 and I44 - Wide number of mutations - Only mildly affected [127]
Mouse ∆Ex45 I44 and I45 - High statistical power - Does not recapitulate the human [125]
∆Ex50 I49 and I50 SpCas9
disease [126]
∆Ex8- - Extensive data/littérature
34 I7 and I34 - Accepted protocols [128]
Humanized ∆Ex45 I44 and I45 (TREAT-NMD). [132,133]
Mouse ∆Ex52 I51 and I52 [131]
∆Ex52 I51 and I52 - Eeasily maintainable [135]
- Severe skeletal/cardiac muscle
Rat phenotype - Higher cost than mice (5×)
∆Ex3- - Allow behavioural experiments
Ex3 and Ex6 SpCas9 - High statistical power [136]
16
Animal

- Severe muscle dystrophy

- Severe cardiomyopathy
Rabbit Ex51 Ex51 SpCas9 - Intermadiate body size - Expensive to maintain
- Lack of accepted protocols [138]
- Relatively short gestational
period
BE3, - Expensive to maintain
Ex13 Ex13 hA3A- - Long gestational period [141]
BE3 - Severe muscular dystrophy
Pig - Accelerated dystrophy compared to
- Anatomy, size and physiology
human
closer to humans - Stress may induced sudden death
Ex27 Ex27 [140]
Ex52 Ex52 - Lack of accepted protocols [139]
- Severe muscular dystrophy
- Expensive to maintain
- Severe cardiomyopathy - High variability
Dog Ex6 Ex6 SpCas9 - Anatomy, size and physiology [146]
- Low statistical power
closer to humans
- Expensive to maintain
Monkey Ex4 or SpCas9 - Long gestational period [147]
Ex4 or Ex46 - Greatest similarity to humans
Ex46 - Recent model, lack of accepted
protocols
Cells 2024, 13, 800 13 of 27

3.2. CRISPR-Based Therapies: From Preclinical to Clinical Trials for DMD

The simplicity, versatility, and precision of CRISPR-/Cas9 offer novel and promising
therapeutic opportunities for DMD. Acting at the DNA level, CRISPR/Cas9 has the poten-
tial to permanently repair the genetic cause of DMD. Here, we report the most advanced
CRISPR/Cas9 therapeutic interventions to correct and restore dystrophin expression, with
a focus on preclinical in vivo studies (Figure 3B).

3.3. NHEJ-Mediated Exon Deletion

More than 60% of DMD patients suffer from exon deletion and 11% of all DMD cases
are due to exon duplications [104,148]. Depending on the genetic defect, deletion of one or
more exons restores the reading frame, resulting in a shorter but functional dystrophin pro-
tein. Many in vitro [149,150] and in vivo studies have demonstrated that the CRISPR/Cas9
system and appropriate pairs of sgRNA can be used to induce exon(s)/intron(s) excision
and restore the DMD reading frame (Figure 3B). In 2016, three independent studies demon-
strated in neonate and adult mdx mice that SpCas9 or SaCas9 and dual sgRNA treatment
delivered by rAAV8, 9 could efficiently delete the mutated exon 23, restore dystrophin
expression and consequently improve muscle morphology, and enhance skeletal and car-
diac muscle function [151–153]. The seminal studies provided evidence for permanent
and efficient gene correction in dystrophic animals. Importantly, such an approach can
be sustained for up to 19 months in murine animals without inducing serious adverse
effects [154–156]. Correction of other DMD genetic defects, including deletions ∆Ex45 [132],
∆Ex52 [157], and ∆Ex52-53 [158,159] or the duplication of Ex18-30 [160], demonstrated
the potential of this CRISPR-based approach to eliminate mutated exon, point mutation,
and exonic duplication in different animal models of DMD, notably in large animal such
as pigs [161] and dogs [162], paving the way for clinical translation. Deletions and du-
plications cluster in two main hotspots in the DMD gene, located at exons 45–55 and
3–9. Following the initial work from Ousterout et al. and the successful transplantation
of CRISPR/Cas9-corrected ∆45-55 patient cells into immunodeficient mice [163], Young
et al. generated a large 45–55 exonic deletion in an hDMD ∆Ex45/mdx mice model [132].
Treatment resulted in the expression of a truncated ∆45-55 dystrophin in muscle animal,
demonstrating the clinical relance of the CRISPR/Cas9 platform. Similarly, Duchêne et al.
demonstrated the restoration of the DMD ∆Ex47-58 reading frame and in vivo dystrophin
expression in hDMD∆Ex52/mdx mice after AAV9 administration delivering SaCas9 and
2 sgRNA targeting the exon 47 and 58 [157]. Whereas such innovative gene editing strategy
is applicable to ~60% of DMD patients, it should be noted that efficiency is correlated
with deletion size and that multi-exon deletions generate a shorter and potentially less
functional dystrophin.

3.4. NHEJ-Mediated Exon Skipping

More than 70% of the DMD population is amendable to exon-skipping strategies
aiming to excise one specific exon to restore a functional open reading frame and produce a
truncated but functional dystrophin protein [164]. Exon-skipping approaches are mutation-
dependent and specific for subsets of the DMD patient population [165]. Disruption of
splicing donor or acceptor sites with a single gRNA and CRISPR/Cas9 introduces InDels,
abolishes splice element function, and leads to the skip of the targeted exon (Figure 3B).
Several in vitro studies in human DMD myoblasts and human iPSCs reported efficient
exon 43, 51, and 53 skipping using this approach [166]. In animal models of DMD, Olson’s
lab induced skipping of exon 51 in neonate ∆Ex50/mdx mice using a dual AAV9 approach
to deliver SpCas9 and three copies of the same gRNA targeting a region adjacent to the
exon 51 splice acceptor site [126]. Systemic treatment conducted a widespread dystrophin
expression in skeletal, respiratory, and cardiac muscles, resulting in histological correction
of the dystrophic defects and significant increased muscle strength. Similar results were
obtained in a humanized ∆Ex50/mdx model [167]. The same group reported similar results
in ∆Ex50-MD dogs, with a restoration of dystrophin levels ranging from 3 to 90% of normal,
Cells 2024, 13, 800 14 of 27

reaching 92% in the heart [162]. Correction of other exon deletions such as ∆Ex43, ∆Ex44,
∆Ex45, and ∆Ex53 were also studied in animal models [125,127,168,169]. Of interest, these
studies demonstrated that the amount of sgRNA delivered to the muscle was critical for
efficient editing in vivo [127] and provided evidence for the long-term benefits of single-cut
gene editing therapy, even under conditions of stress [168]. Compared with CRISPR-
mediated exon deletion using two sgRNA, single-cut exon skipping is a safer approach as
it reduces potential off-target effects and DNA damage. Importantly, such exon-skipping
strategies may also result in exon reframing.

3.5. NHEJ-Mediated Exon Reframing

Similarly to single-cut exon skipping, the exon reframing approach relies on the use
of a single gRNA targeting a frameshift mutation and the subsequent generation of small
InDels by NEHJ to restore a functional reading frame. In theory, small indels generated up-
stream of a deleterious mutation have a 1 in 3 probability of restoring the reading frame. If
successful, this approach will restore a larger dystrophin protein compared with exon skip-
ping (Figure 3B). Several in vitro studies [163,170] demonstrated the efficacy of this CRISPR
strategy. Positive results were observed in vivo in ∆Ex50/mdx mice after AAV9 delivery
of CRISPR components to restore the open reading frame of exon 51. Muscle function im-
proved toward wildtype levels even with low gene editing efficiency (8–12%), resulting in
27–45% of dystrophin expression in skeletal, respiratory, and cardiac muscles [171]. This ap-
proach was applied to several mdx mice, including mEx23 [130], ∆Ex50 [126], h∆Ex43 [125],
h∆Ex44 [127], h∆Ex45 [125], and h∆Ex52 [125] mdx mice. Interestingly, Min et al. compared
both exon skipping and reframing strategies for three DMD mutations and concluded that
exon reframing was more efficient than exon skipping in restoring dystrophin [125].

3.6. HDR-Mediated Gene Correction

Unlike NHEJ-based strategies, HDR-mediated gene editing has the potential to restore
the full-length dystrophin and to correct mutations in the N- and C-terminal region of the
protein (Figure 3B). The first in vivo proof-of-concept of this approach was provided in
2014 [172] by zygotic injection of SpCas9/sgRNA and an exogenous DNA template in mdx
mouse embryos. This seminal work demonstrated that ~17% of gene editing was sufficient
to restore dystrophin expression to wild-type levels. Consequently, several investigations
have attempted to correct mutations in exons 23 [173,174], 44 [170], and 53 [158] of the
Dmd gene using similar knock-in approaches. In mdx4cv , intramuscular injection of an
AAV6 delivering Cas9/sgRNA and a donor template rescued dystrophin production
to 1.8–8.4% of the wild-type level following a 0.18% gene editing efficiency [158]. In a
GRMD dog model, intramuscular injection of Cas9 components and a corrective ssODN
restored dystrophin expression to 6–16% of normal levels [175]. The NHEJ-based knock-in
homology-independent targeted integration (HITI) approach has also been leveraged to
correct genetic defects [176,177]. By including two Cas9 cleavage sites flanking the donor
sequence, HITI allows efficient integration of a DNA template at the targeted genomic
site. Pickar-Oliver et al. successfully knocked-in exon 52 and super-exon 52–79 in an
hDMD∆52 mouse model [131]. Despite the low efficiency, these results demonstrated
that HITI can restore the dystrophin protein in vivo. While the accuracy of HRD- and
HITI-based strategies is appealing, the major limitation of these approaches is their low
efficiency. Additionally, inverted/unwanted integration of the DNA donor may occur,
resulting in potential alteration of dystrophin expression. Extensive optimization and safety
studies will be required to advance these therapies to the clinic.

3.7. Base Editing

Beyond traditional NHEJ- and HDR-based CRISPR/Cas9 systems, generation of
catalytically impaired Cas9 (D10A) fused to a base-modifying enzyme introduced the first
iteration of a more precise and safer gene editor [23] (Figure 2B). Cytosine base editors
(CBEs) [19] and adenine base editors (ABEs) [18] respectively allow C:G to T:A and A:T to
Cells 2024, 13, 800 15 of 27

G:C base pair transitions without DSBs, DNA donor template, or HDR, offering a promising
editing avenue to precisely correct single point and non-sense mutations (Figures 1 and 2),
accounting for ~27% of the DMD cases [178]. Described as safer than CBEs due to a higher
specificity and lower off-target activity [179,180], several ABE systems were successfully
used in different mouse models of DMD (Figure 3B). Ryu et al. used trans-splicing to
deliver a full ABE with two AAV9s to correct a point mutation generating a premature
stop codon in exon 20 of a murine Dmd gene [181]. Eight weeks post infusion, dystrophin
expression was detected in 17% of the skeletal myofibers. Similarly, Hu and colleagues
developed a single AAV9-iABE-NGA systemic treatment designed to correct a non-sense
mutation in exon 53 of the mdx4cv mouse [182]. A widespread restoration of dystrophin
expression and subsequent histopathological and functional benefits were noted 10 weeks
post-injection. Importantly, therapeutic benefits were maintained 10 months after injection,
with a near complete rescue of dystrophin in the heart and 15% rescue in skeletal muscle
fibers. By altering splice sites, BEs can promote exon skipping and correct mutations and
deletions in the DMD gene. Systemic administration of AAV9s delivering CBE and sgRNA
designed to skip exon 4 in DmdE4 mice resulted in efficient dystrophin restoration and
rescue of muscular dystrophy phenotype [183]. Similar results were obtained by Chemello
and al. following intramuscular injection of dual AAV9s encoding ABE components as a
split-intein trans-splicing system promoting exon-50 skipping in ∆Ex51 mice [184].
While these preclinical studies demonstrate feasibility and efficacy of BEs for many
DMD mutations, delivery remains a major challenge. Due to their large size, BEs require
dual-vector delivery and increased viral doses associated with immunological and toxico-
logical concerns. A novel optimized all-in one BE solution able to fit into a single AAV [185]
may lead to further the preclinical development of these innovative therapies for DMD.

3.8. Prime Editing

The versatility of prime editing has opened up an attractive horizon for the treatment
of various genetic defects such as DMD [186] (Figures 2C and 3B). Olson’s lab applied first
prime editing to ∆Ex51 DMD patient-derived iPSCs to reframe exon 52 and successfully
restored dystrophin expression to 24.8–39.7% of healthy levels [184]. Similarly, Tremblay’s
lab used PE to induce substitutions, insertions, and deletions in the splice donors for exons
51 and 53 to correct frameshift mutations in the DMD gene carrying exon 52 or exons
45 to 52 deletions [187] and to correct point mutation in the DMD gene [121,188]. Recently,
Anzalone et al. demonstrated that twin PE systems can precisely excise exon 51 in the
dystrophin gene in vitro [28], paving the way for an innovative gene editing solution
for DMD.
While this technology has the potential to correct a large variety of DMD mutations,
further in vivo applications are required to progress and translate this approach to pa-
tients and, similarly to base editing, size constraints remain a challenge to be solved for
in vivo delivery.

3.9. CRISPR/Cas9 Activation

In addition to enabling the engineering of eukaryotic genomes, recent alterations to
the CRISPR/Cas9 system have provided opportunities for regulating gene expression [189]
(Figure 2D). Modulation of disease-modifier genes can provide therapeutic solutions ap-
plicable to all DMD patients irrespective of their dystrophin mutation [115,190]. Utrophin
(UTRN), an autosomal and functional paralogue of dystrophin expressed at the sarcolemma
of regenerated myofibers [191,192], has the potential to compensate for the primary defect
in DMD [193]. Seminal preclinical studies demonstrate that expression of UTRN suppresses
functional signs of dystrophinopathy in a dose dependent manner [194] without toxic-
ity [195], and strongly support UTRN as a functional surrogate for dystrophin [190,196].
Using CRISPRa (dCas9-VP160) targeted to the UTRN promoter, Wojtal et al. successfully
modulated UTRN expression in DMD myoblasts [197]. Liao and colleague demonstrated
a three- four-fold in vivo increase in UTRN levels in mdx mice using a different CRISPRa
Cells 2024, 13, 800 16 of 27

system [198] (Figure 3B). While these preclinical studies emphasize the potential of CRISPR-
based UTRN upregulation for DMD, such approaches require long-term efficacy and
toxicology in vivo assessment.
Recently, Cure Rare Disease, Inc. launched the first CRISPR-based clinical trial for
DMD (NCT05514249) [199]. The n-of-1 clinical trial involved a single 27-year-old DMD
patient suffering from an exon 1 deletion, lacking the dystrophin muscle isoform (Dp427m).
Phase 1 aimed to assess the safety and preliminary efficacy of CRD-TMH-001, an AAV9-
dCas9-VP64 delivered intravenously at a 1×1014 vg/kg dose and designed to upregulate
cortical dystrophin through the conserved Dp427p promoter. Six days after receiving
the therapy, the patient showed signs of mild cardiac dysfunction and pericardial effu-
sion, leading to cardiac arrest two days later. Post-mortem examination revealed severe
acute-respiratory distress syndrome (ARDS) with diffuse alveolar damage. Investigators
concluded that the high dose of AAV triggered the adverse event and not the CRISPR
therapy itself. Despite remarkable success in gene therapy, lethal immunotoxicity in high-
dose systemic AAV therapy was previously reported in treated DMD, SMA, and XLMTM
patients [200]. This first reported case of severe ARDS linked to an AAV product provided
a painful but valuable lesson that will help de-risk the translation of gene therapies for
future patients and remind us of the challenges we still need to overcome.

4. From the Same Technology to Different Stages: A Comparison of CRISPR-Based

Strategies for Blood and Muscle Fields
While CRISPR/Cas9 strategies for blood disorders rapidly advanced from early
in vitro studies to clinical trials, gene editing for neuromuscular diseases has not yet
reached similar milestones. Clinical translation of the same technology may progress
differently across medical fields because of disease-specific limitations.
While hematopoietic stem cells can be purified, corrected ex vivo, and re-infused into
the patient [201], CRISPR/Cas9 components need to be systemically delivered in order
to reach the muscles. For the treatment to be effective, AAV vectors carrying the editing
machinery must transduce skeletal, respiratory, and cardiac muscle tissues specifically and
efficiently. This requires the choice of adequate serotypes and appropriate dosing, balancing
efficacy considerations and risks associated with systemic AAV vector delivery [202]. From
the administration route to vector manufacturing, neuromuscular diseases currently face a
higher level of complexity compared with blood disorders, explaining why CRISPR/Cas9
therapies have not yet reached the same clinical stages. While extensive optimization efforts
are currently ongoing to address such challenges [202], it should be noted that DMD will
greatly benefit from the clinical success of CRISPR in blood disorders and future long-term
follow-up studies.
A major bottleneck to clinical translation for novel DMD therapies is the lack of
suitable animal models. The widely used mdx mouse [124] does not recapitulate the human
disease and does not offer the appropriate genetic context for sequence-based approaches,
such as exon skipping, strop codon read through, and CRISPR/Cas9 strategies. CRISPR
has been an incredible tool to produce novel humanized animal models for DMD, speeding
up the translation of sequence-based approaches to patients.

5. Conclusions: Challenges and Current Trends

In recent years, CRISPR has emerged as a revolutionary technology with the potential
to permanently and precisely edit any DNA locus and correct genetic errors. From CRISPR-
Cas to base and prime editors, the CRISPR tool box has generated innovative preclinical
models and corrective strategies for many genetic disorders, bringing more breakthrough
therapies closer to the patients. The last decade has witnessed the rapid development of
this technology for monogenic blood disorders, from the early proof-of principle studies
to the first regulatory approval of a CRISPR-based therapy for SCD and TDT. To date,
there are currently 94 clinical trials for hereditary and non-hereditary diseases based on
CRISPR technologies ([Link] Nevertheless, as illustrated by blood
Cells 2024, 13, 800 17 of 27

and neuromuscular disorders, there are still many hurdles to overcome to expand clinical
applications of gene editing therapies, including long-term safety and off-target activity
and also cost, accessibility, and manufacturing. Some concerns are specific to the targeted
organ system.
Despite the success of Casgevy in SCD and TDT and many promising results from
undergoing clinical trials, we still do not know the long-term efficacy and safety of these
treatments. While effective at reducing vaso-occlusive events and transfusion dependency
in SCD, we need long-term follow-up studies to establish whether Casgevy can also de-
crease disease burden in other organs like heart, lung, and kidney. These studies will also
evaluate the persistence of high HbF expression over time, a parameter that is particularly
critical for SCD patients, as levels below 20% are not considered protective. HSPCs con-
stantly replenish all blood cell types through repeated cell divisions and differentiation
steps. By editing HSPCs, the DNA modifications will be inherited by all the HSPC-derived
blood lineages. DNA damage and DNA DSBs can lead to genotoxic effects [13–16] and
loss of self-renewal in HSPCs. Moreover, unintended modifications can occur when sim-
ilar DNA sequences are targeted, leading to the generation of off-target mutations that
can potentially have deleterious consequences or provide selective advantages to specific
blood lineages. The progressive transition to DSB-free CRISPR systems such as base and
prime editing and the development of high-fidelity Cas enzymes with lower off-target
activity [203] will help reduce the mutational risk. So far, no evidence of DSB-associated
genotoxicity has been observed in patients receiving Casgevy. It might still be too early
to rule out long-term genotoxicity, as two patients who underwent gene therapy for SCD
developed blood malignancies 3–5.5 years post-transplant [204]. Clonal expansion has
been observed in the patient who participated in the Graphite Bio trial; however, the
clinical significance of this event remains unclear. Developing ways to reduce, detect, and
predict genotoxicity of CRISPR-based therapies remains essential to safe clinical implemen-
tation. Most CRISPR-based therapies for genetic blood disorder require HSPC mobilization
and myeloablation, a procedure associated with serious complications. Ex vivo genetic
modification and transplantation of HSPCs is costly and can only be performed by special-
ized treatment centers with highly trained personnel, allowing a small portion of eligible
patients to be treated each year. Moreover, since Casgevy could cost as much as USD
2 million per treatment, it would be practically inaccessible to low-income countries where
the majority of TDT and SCD patients live [205].
Neuromuscular disorders cannot be corrected ex vivo and therefore rely on systemic
viral delivery of the editing machinery, raising immunogenicity concerns both for the high
AAV doses required and the long-term expression of CRISPR components [166]. Despite
high transduction efficiency, durable therapeutic benefits, and remarkable clinical successes,
the AAV platform suffers from several limitations. First, the limited cargo capacity limits
the delivery options of CRISPR/Cas9 components, too large to be expressed from a single
AAV vector. While dual-AAV systems are often used, all-in-one constructs [171] with
truncated Cas9 nickase represent attractive solutions that are currently under investigation
in vivo. The high dose required for therapeutic use has been previously associated with im-
munotoxicity in AAV-treated patients [200], and is a hurdle to manufacturing. Pre-existing
immunity to AAVs and CRISPR components might limit access to therapies and will require
careful monitoring and intervention pre- and post-infusion [206]. Optimized forms of Cas9
with modified epitopes hold great promise in reducing host immunogenicity [207]. In
addition, the development of innovative AAV serotypes offering greater efficiency at lower
doses [205] may overcome, in part, these challenges. Non-viral solutions such as gold and
lipid nanoparticles that deliver mRNA to HSPC [203] and other tissues in vivo [204] may
also play an important role in delivering CRISPR/Cas systems in the future. Alongside
all these considerations, the durability of CRISPR/Cas9 treatment is pivotal for DMD and
other muscle disorders. The efficient delivery of CRISPR/Cas9 elements to satellite cells
may ensure long-lasting therapeutic benefits in growing patients. Whereas preclinical stud-
ies showed that systemic AAV CRISPR delivery can target satellite cells and editing persist
Cells 2024, 13, 800 18 of 27

up to 18 months, efficiency needs to be improved and further optimization is required to

target this cell population.
The last decade witnessed the great potential of CRISPR/Cas9 systems, however exten-
sive preclinical and clinical investigations are still needed to overcome several therapeutic
challenges. Lessons learned from these studies and future CRISPR-based research in the
hematological and neuromuscular fields will further the development of novel therapies
for other devastating genetic diseases.

Author Contributions: Conceptualization, G.P. and S.G.; Methodology, M.L., M.G., G.P. and S.G.;
Investigation, M.L., M.G., G.P. and S.G.; Data curation, M.L., M.G., G.P. and S.G; Writing—original
draft preparation, G.P. and S.G.; Writing—review and editing, M.L., M.G., G.P. and S.G.; visualization,
M.L., M.G., G.P. and S.G.; Supervision, G.P. and S.G.; Project administration, S.G.; Funding acquisition,
G.P. and S.G. All authors have read and agreed to the published version of the manuscript.
Funding: M.G. and S.G. are funded by SQY Therapeutics. M.L. is funded by Genethon. G.P. is funded
by Children’s Hospital of Philadelphia (CHOP) and the CHOP Cell and Gene Therapy Seed Grant.
Conflicts of Interest: Authors Marine Geoffroy and Simon Guiraud were employed by the company
SQY Therapeutics. The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential conflict of interest. The
SQY Therapeutics had no role in the design of the study; in the collection, analyses, or interpretation
of data; in the writing of the manuscript, or in the decision to publish the results.

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