Nanoparticles targeting hematopoietic stem and progenitor cells: Multimodal carriers for the treatment of hematological diseases

Author: CAST

Publisher: Zenodo

DOI: 10.3389/fgeed.2022.1030285

Source: https://zenodo.org/records/17311097/files/35_fgeed-04-1030285.pdf

Nanopa icles a ge ing
hema opoie ic s em and
p ogeni o cells: Mul imodal
ca ie s o he ea men o
hema ological diseases
Luis J. C uz
1
, Somayeh Rezaei
1
, F ank G os eld
2
,
Sjaak Philipsen
2
and Ch is ina Eich
1
*
1
T ansla ional Nanobioma e ials and Imaging, Depa men o Radiology, Leiden Uni e si y Medical
Cen e , Leiden, Ne he lands,
2
E asmus Uni e si y Medical Cen e , Depa men o Cell Biology,
Ro e dam, Ne he lands
Mode n-day hema opoie ic s em cell (HSC) he apies, such as gene he apy,
modi y au ologous HSCs p io o e-in usion in o myelo-condi ioned pa ien s
and hold g ea p omise o ea men o hema ological diso de s. While his
app oach has been success ul in nume ous clinical ials, i elies on
ansplan a ion o ex i o modiﬁed pa ien HSCs, which p esen s se e al
limi a ions. I is a cos ly and ime-consuming p ocedu e, which includes
only ew pa ien s so a , and ex i o cul u ing nega i ely impac s on he
iabili y and s em cell-p ope ies o HSCs. I i al ec o s a e used, his
ca ies he addi ional isk o inse ional mu agenesis. A he apy deli e ed o
HSCs in i o, wi h minimal dis u bance o he HSC niche, could o e g ea
oppo uni ies o no el ea men s ha aim o e e se disease symp oms o
hema opoie ic diso de s and could b ing sa e, e ec i e and a o dable gene ic
he apies o all pa s o he wo ld. Howe e , subs an ial unme needs exis wi h
espec o he in i o deli e y o he apeu ics o HSCs. In he las decade, in
pa icula wi h he de elopmen o gene edi ing echnologies such as CRISPR/
Cas9, nanopa icles (NPs) ha e become an eme ging pla o m o acili a e he
manipula ion o cells and o gans. By employing su ace modiﬁca ion s a egies,
di e en ypes o NPs can be designed o a ge speciﬁc issues and cell ypes in
i o. HSCs a e pa icula ly di ﬁcul o a ge due o he lack o unique cell su ace
ma ke s ha can be u ilized o cell-speciﬁc deli e y o he apeu ics, and hei
shielded localiza ion in he bone ma ow (BM). Recen ad ances in NP
echnology and gene ic enginee ing ha e esul ed in he de elopmen o
ad anced nanoca ie s ha can deli e he apeu ics and imaging agen s o
hema opoie ic s em- and p ogeni o cells (HSPCs) in he BM niche. In his
e iew we p o ide a comp ehensi e o e iew o NP-based app oaches
a ge ing HSPCs o con ol and moni o HSPC ac i i y in i o and in i o,
and we discuss he po en ial o NPs o he ea men o malignan and non-
malignan hema ological diso de s, wi h a speciﬁc ocus on he deli e y o gene
edi ing ools.
OPEN ACCESS
EDITED BY
Yizong Hu,
Johns Hopkins Uni e si y, Uni ed S a es
REVIEWED BY
Ma ia Rosa Lidonnici,
San Ra aele Tele hon Ins i u e o Gene
The apy, I aly
S ujan Ma epally,
Cen e o S em Cell Resea ch, India
*CORRESPONDENCE
Ch is ina Eich,
[email p o ec ed]
SPECIALTY SECTION
This a icle was submi ed o Genome
Edi ing Tools and Mechanisms,
a sec ion o he jou nal
F on ie s in Genome Edi ing
RECEIVED 28 Augus 2022
ACCEPTED 10 Oc obe 2022
PUBLISHED 02 No embe 2022
CITATION
C uz LJ, Rezaei S, G os eld F, Philipsen S
and Eich C (2022), Nanopa icles
a ge ing hema opoie ic s em and
p ogeni o cells: Mul imodal ca ie s o
he ea men o
hema ological diseases.
F on . Genome Ed. 4:1030285.
doi: 10.3389/ geed.2022.1030285
COPYRIGHT
© 2022 C uz, Rezaei, G os eld,
Philipsen and Eich. This is an open-
access a icle dis ibu ed unde he
e ms o he C ea i e Commons
A ibu ion License (CC BY). The use,
dis ibu ion o ep oduc ion in o he
o ums is pe mi ed, p o ided he
o iginal au ho (s) and he copy igh
owne (s) a e c edi ed and ha he
o iginal publica ion in his jou nal is
ci ed, in acco dance wi h accep ed
academic p ac ice. No use, dis ibu ion
o ep oduc ion is pe mi ed which does
no comply wi h hese e ms.
F on ie s in Genome Edi ing on ie sin.o g01
TYPE Re iew
PUBLISHED 02 No embe 2022
DOI 10.3389/ geed.2022.1030285
KEYWORDS
hema opoie ic s em cells, nanopa icles, a ge ing, gene he apy, imaging, deli e y
1 In oduc ion
1.1 Scope o his e iew
Hema opoie ic s em cells (HSCs) ha e he capaci y o eplenish
all blood cell lineages du ing he s eady-s a e cellula u no e o he
blood sys em, and unde s ess condi ions such as acu e
inﬂamma ion o HSC mobiliza ion (O kin and Zon, 2008;Sun
e al., 2014;Rod iguez-F a icelli e al., 2018). Dis inc lineage-
commi ed hema opoie ic p ogeni o cells (HPCs) eme ge om
an indi idual HSC h ough many di e en ia ion s eps and cell
di isions, while HSCs a e also main ained h ough sel - enewal
(O o d and Scadden, 2008;No a e al., 2016;Vel en e al., 2017;
Rod iguez-F a icelli e al., 2018). He edi a y hema ological
diso de s (including hemophilia, blood clo ing diso de s,
halassemias and sickle cell disease) and acqui ed diso de s
(including myelodysplas ic synd omes and malignancies such as
lymphomas, leukemias and myelomas) a e examples o pa hologies
a ec ing he hema opoie ic sys em, many o which a e caused by
mu a ions (Bao e al., 2019). One way o ea hese diso de s is
eplacemen o diseased HSCs by heal hy allogeneic HSCs
(Gyu kocza e al., 2010). HSC ansplan a ion is one o he
majo medical disco e ies o he 20 h cen u y and has been used
o o e 50 yea s o he ea men o leukemias and monogenic
blood- ela ed diseases (Copelan, 2006;Appelbaum, 2007). While
HSC ansplan a ions sa e ens o housands o li es pe yea
wo ldwide (G a wohl e al., 2010), many pa ien s emain
dep i ed o his li e-sa ing p ocedu e due o he lack o a
compa ibledono o aninsu ﬁcien numbe o HSCs in he
g a . This can lead o ea men - ela ed mo bidi y and
mo ali y (Mimeaul e al., 2007). Al e na i ely, epai o
disease-causing genes in au ologous HSCs ex i o ollowed
by HSC ansplan a ion o di ec ea men in i o migh be
he “holy g ail” o malignan and non-malignan
hema ological diseases, p o ided ha su ﬁcien numbe s o
au ologous HSCs could be co ec ed. In he las decade, in
pa icula wi h he de elopmen o gene edi ing echnologies
such as CRISPR/Cas9, nanopa icles (NPs) ha e become an
eme ging pla o m o acili a e he gene ic manipula ion o
cells and issues in i o and in i o. NPs a e submic on-sized
pa icles ha can be gene a ed om a a ie y o componen s,
including polyme s, lipids, me als and a e ea h elemen s, o
can be isola ed as ex acellula esicles (EVs) om cells o
assembled om i us capsids (Figu e 1). NPs ep esen
p omising ools o moni o ing and con olling HSC
ac i i y in i o due o hei capaci y o p o ec a payload
om p ema u e deg ada ion and media e endosomal escape o
enable nucleic acid and Cas9 ansloca ion o he cy oplasm
and nucleus (Yin e al., 2014), while ci cum en ing e ﬁcacy
and sa e y issues o classical i al ehicles. Mo eo e , ecen
FIGURE 1
Schema ic ep esen a ion o di e en ypes o nanopa icles commonly used o deli e he apeu ics and imaging agen s in biomedical
applica ions.
F on ie s in Genome Edi ing on ie sin.o g02
C uz e al. 10.3389/ geed.2022.1030285
de elopmen s in nano echnology ha e demons a ed he
easibili y o si e-speciﬁc deli e y by sma polyme s
ea u ing spa io empo al elease kine ics (Zhuo e al., 2021),
which could acili a e he manipula ion o HSCs in si u while
limi ing o - a ge deli e y.
Se e al NP o mula ions ha e been app o ed o clinical use,
mos ly as a deli e y ehicle o he apeu ics in he ﬁeld o cance
and egene a i e medicine, as a deli e y pla o m o medical
imaging agen s, o as a accine in he ﬁeld o in ec ious diseases
(Anselmo and Mi ago i, 2019). NP-based diagnos ics and
he apies ha e also ecei ed conside able a en ion in he ﬁeld
o hema ological diso de s, such as o he de ec ion o
ci cula ing umo cells by an i-CD20-coa ed quan um do s
(Sha ia i a e al., 2019), o he ea men o anemia by o ally
adminis e ed i on-based NPs o he manipula ion o
hema opoie ic s em- and p ogeni o cells (HSPCs) in he e al
and adul hema opoie ic niche (Za iwala e al., 2013;Hosny e al.,
2015).
In his e iew, we p o ide a comp ehensi e o e iew o NP-
based app oaches a ge ing HSPCs in biomedical applica ions. In
he ﬁ s pa , we ocus on how HSPCs in e ac wi h NPs, aking
in o accoun he speciﬁc biology o HSCs and HPCs, and hei
localiza ion in hema opoie ic niches. In he second pa , we
e iew he use o NPs o con ol and moni o HSPC ac i i y
in i o and in i o. In he hi d pa o his e iew, we discuss he
po en ial o NPs o he ea men o malignan and non-
malignan hema ological diso de s, wi h a speciﬁc ocus on
he deli e y o gene edi ing ools.
1.2 Nanopa icles
NPs a e widely accep ed o ha e a size be ween 5–300 nm
(al hough s uc u es o up o 1,000 nm ha e also been
epo ed). Based on hei chemical composi ion, hey a e
commonly g ouped in o ca bon-based NPs (ca bon
nano ubes and ulle enes), ino ganic NPs (quan um do s,
me allic NPs, a e ea h-ma e ial NPs) and o ganic NPs
(lipid NPs, polyme ic NPs and EVs) (Figu e 1). The use o
NPs as d ug deli e y sys em has many ad an ages o e he
deli e y o naked d ugs: 1) due o hei la ge inne olume,
NPs can be loaded wi h hyd ophilic and hyd ophobic
compounds, including ﬂuo opho es, me als, pep ides,
p o eins, nucleic acids o biomime ic molecules, which can
inc ease he concen a ion o hese compounds locally; 2)
d ug encapsula ion in NPs can imp o e he
biocompa ibili y and s abili y o con en ional d ugs and
o e come p oblems o insolubili y; 3) in con as o
con en ional d ugs, NPs p esen an enhanced ci cula ion
ime in he blood s eam; 4) NPs can be designed o be
mul i unc ional by exe ing bo h diagnos ic and he apeu ic
ac ions; and 5) he NP su ace can be unc ionalized wi h
a ge ing moie ies o pe mi si e- and/o cell-speciﬁc
payload deli e y and imp o e he a io o e ﬁcacy/
cy o oxici y o he encapsula ed payload. This educes
ad e se side e ec s o en associa ed wi h sys emically
applied high doses o d ugs.
1.3 Nanopa icle up ake
NP up ake is inﬂuenced by h ee main ac o s: 1) The
physicochemical p ope ies o NPs, such as size, polydispe si y
index (measu e o he he e ogenei y o a sample based on size),
shape, cha ge, su ace modiﬁca ion and su ace hyd ophobici y/
hyd ophilici y; 2) The physiological p ope ies o a ge cells and
hei mic oen i onmen (e.g., p esence o cell su ace
p o eoglycans o ecep o s, le els o se um p o eins); and 3)
expe imen al ac o s, including empe a u e, incuba ion ime,
osmola i y and ionic s eng h (He e al., 2021).
A e encoun e wi h he cell memb ane, NPs a e aken up
ia he cellula endocy osis machine y by wo main mechanisms,
phagocy osis and pinocy osis. Phagocy osis is he p e e ed
up ake mechanism o la ge pa icles (>500 nm), such as
pa hogens, cell agmen s, o NPs. Pinocy osis (including
mac opinocy osis, cla h in-media ed endocy osis, ca eolae-
media ed endocy osis and cla h in- and ca eolin-independen
endocy osis) is ega ded as he dominan mechanism o he
up ake o NPs o less han 500 nm (Zhao and S enzel, 2018).
Posi i ely cha ged NPs a e in e nalized apidly ia he cla h in-
media ed pa hway, while nega i ely cha ged NPs a e in e nalized
mainly h ough pa hways o he han cla h in and ca eolin
(Ha ush-F enkel e al., 2007). Howe e , posi i ely cha ged
NPs a e in gene al associa ed wi h highe cy o oxici y
(Goodman e al., 2004;Oh e al., 2010), hus as up ake a es
a e no necessa ily beneﬁcial. Cu en NP-based app oaches
a ge ing HSCs in biomedical applica ions ocus a ound h ee
majo objec i es (Figu e 2). Fi s ly, imp o ing labelling s a egies
o moni o ansplan ed HSCs by di e en imaging modali ies.
Secondly, he deli e y o d ugs and gene edi ing ools o modula e
HSCs and he BM niche o he de elopmen o human
he apeu ics, and hi dly, undamen al esea ch o de elop
no el ools o a ge and ack NPs biodis ibu ion in i o.
Examples o NPs ha ha e been used o a ge HSPCs o
he apy o moni o ing pu poses mainly belong o he g oup
o o ganic and ino ganic NPs (Table 1).
1.4 Hema opoie ic s em cells and
hema opoie ic p ogeni o cells
HSCs lack known unique cell su ace ma ke s ha can be
used o s aigh o wa d cell isola ion. Ins ead, CD34 is
commonly used o s udy HSPCs in he labo a o y and o
en ichmen p io o BM ansplan a ion (Su he land e al.,
1990;Baum e al., 1992). CD34 is a ansmemb ane
F on ie s in Genome Edi ing on ie sin.o g03
C uz e al. 10.3389/ geed.2022.1030285
glycop o ein exp essed on HSPCs and many ascula endo helial
cells (ECs). Thus, in addi ion o a ew HSCs he CD34
+
ac ion
includes ECs and imma u e and ma u e HPCs ha can be u he
dis inguished by addi ional ma ke s (Doula o e al., 2012).
P og ess has been made in cha ac e izing human long- e m
epopula ing HSCs (LT-HSCs) based on eng a men analysis
o HSC popula ions as CD19
−
CD34
+
CD38
−
CD45RA-
CD49 +CD90
+
(Thy-1) (No a e al., 2016). Index-so ing in
combina ion wi h RNA sequencing u he e ealed ha
especially he CLEC9A
h
iCD34
lo
subse is en iched in LT-
epopula ing HSCs (Belluschi e al., 2018). O no e, di e en
ma ke s ha e been employed in s udies in human
(CD34
+
CD90
+
CD133+ (Wang e al., 2015;Schi oli e al., 2019;
Fe a i e al., 2020) and CD34
+
CD90
+
(Michalle e al., 2000;
Neg in e al., 2000)) e sus non-human p ima es
(CD34
+
CD90
+
CD45RA (Rad ke e al., 2017;Humbe e al.,
2019). As mos s udies on NPs and HSCs we e conduc ed
using CD34
+
HSPCs, cau ion needs o be aken ega ding he
in e p e a ion o NP a ge ing da a owa ds HSCs. In his e iew,
we will e e o CD34
+
cells as HSPCs, unless s a ed o he wise.
In con as o HPCs, HSCs a e p ima ily main ained in a
quiescen (G0) s a e in specialized BM niches (Zhang e al., 2003).
This s a e is accompanied by speciﬁc physiological p ope ies,
such as cell cycle a es , educed ansc ip ional and ansla ional
ac i i y and unique ene gy me abolism (Passegué e al., 2005;
Takubo e al., 2013;Yu e al., 2013;I o e al., 2016) and
FIGURE 2
O e iew o NPs a ge ing HSPCs in bioimaging, undamen al esea ch and he de elopmen o human he apeu ics. De elopmen o no el NP
designs and bioconjuga ion s a egies enables he applica ion o mul i unc ional NPs o in in i o imaging and he apy o HSPCs.
F on ie s in Genome Edi ing on ie sin.o g04
C uz e al. 10.3389/ geed.2022.1030285
TABLE 1 Summa y o NP sys ems a ge ing HSPCs and he BM in i o and in i o.
Ca ego y NPs Size (nm) Ta ge Pu pose Re
Polyme ic
NPs
Polys y ene and PLLA NPs 116–131 Human HSPCs S udy he inﬂuence o NPs on
cell di e en ia ion capaci y
and unc ionali y
B üs le e al. (2015)
Ca boxyla ed polys y ene NPs 40 Human HSPCs S udy NP loading beha io De ille e al. (2017)
Chi osan NPs 200–700 Mu ine BM-de i ed
HSPCs
S udy he inﬂuence o NP size
on cell iabili y and
unc ionali y
Zaki e al. (2015)
Chi osan NPs 200 Human pe iphe al
blood-de i ed HSPCs
S udy how deace yla ion
deg ee and molecula weigh o
chi osan a ec cell iabili y and
unc ionali y
Jesus e al. (2020)
PLGA-NPs encapsula ing ﬂuo ine 19 (
19
F) 290 Human co d blood
-de i ed HSPCs
Cell labeling and MR imaging Duinhouwe e al.
(2015)
P o amine sul a e-modiﬁed PLGA-NPs
encapsula ing pe ﬂuo o-1,5-c own e he
210 Human HSPCs Cell labeling and MR imaging Aday e al. (2014)
PLGA NPs encapsula ing Wn 3a p o ein 178 ESCs Deli e y and s abiliza ion o
Wn 3a p o ein
Tuysuz e al. (2017)
Chi osan/ ipolyphospha e/ ucoidan NPs
encapsula ing SDF-1
173–403 BM-MSCs Deli e y o SDF-1 Huang and Liu, (2012)
Alend ona e-modiﬁed PLGA-PEG-NPs
encapsula ing bo ezomib
150–200 Bone D ug deli e y o BM Swami e al. (2014)
T iblock co-polyme Poloxame -40-
modiﬁed polys y ene NPs
60, 150, 250 Rabbi bone
ma ow (BM)
Ta ge ing he BM Po e e al. (1992)
PLGA-NPs encapsula ing CRISPR RNPs 300–400 Human HSPCs Deli e y o CRISPR RNPs o
edi he β-globin gene locus
C uz e al. (2021)
Poly-β-amino es e NPs encapsula ing
CRISPR RNPs
200 GCSF-mobilized
human CD34
+
HSPCs
Deli e y o CRISPR RNPs o
edi he CD33 gene locus
El-Kha ag e al. (2022)
PLGA-NPs encapsula ing PNA and
DNA NPs
150 HSCs In i o gene edi ing o he
CCR5 and β-globin gene loci
McNee e al. (2011);
McNee e al. (2013)
Memb ane glycan-modiﬁed ca boxyla ed
polys y ene NPs
40, 100, 200 Human HSPCs S udy he inﬂuence o NP size
and memb ane-associa ed
glycans on NP loading
beha io
Wa hiong e al. (2019)
Lipid-
based NPs
Liposomes encapsula ing USPIO (P7228) 20–50 Human co d blood-
de i ed HSPCs
MR imaging o p elabeled
HSPCs in he BM
Dald up-Link e al.
(2005)
Liposomes encapsula ing Wn 3a p o ein 130–150 ESCs Deli e y and s abiliza ion o
Wn 3a p o ein
Tuysuz e al. (2017)
Maleimide headg oup-modiﬁed liposomes
and liposome-like syn he ic NPs
encapsula ing GSK-3βinhibi o
230 Conjuga ed o mu ine
HSCs
Cell enginee ing/adju an
deli e y o imp o e ou come
o HSC ansplan a ion
S ephan e al. (2010)
PEG-lipid NPs encapsula ing
siRNA (“BM1”)
60–80 Mu ine BMECs Gene silencing in BMECs Sago e al. (2018)
Lipid–PEG NPs encapsula ing small siRNA
(NicheEC-15″)
60–80 Mu ine BMECs Gene silencing in BMECs K ohn-G imbe ghe
e al. (2020)
Alend ona e-modiﬁed liposomes
encapsula ing SDF-1 gene
116, 123 Bone/Os eoblas s Bone a ge ed plasmid deli e y
o ec opic gene exp ession
Chen e al. (2018)
Lipid NPs encapsula ing Cas9 mRNA and
sgRNAs
Human HSCs Gene edi ing o BM cells In ellia The apeu ics, I.,
2021
RGD-PEG- modiﬁed liposomes
encapsula ing siRNA o doxo ubicin
100–210 ECs SiRNA deli e y o ECs Schi ele s e al. (2005)
Mul ilamella lipid esicles encapsula ing
GSK-3βinhibi o
496 Conjuga ed o mu ine
HSCs
Cell enginee ing/adju an
deli e y o enhance
p oli e a ion kine ics o in
u e o ansplan ed HSCs
Loukogeo gakis e al.
(2019)
PLGA NPs encapsula ing γPNAs and
dono DNA
200 Mu ine HSPCs In u e o deli e y o PNAs and
dono DNAs o co ec a
Riccia di e al. (2018)
(Con inued on ollowing page)
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C uz e al. 10.3389/ geed.2022.1030285

dis inguishes HSCs om mo e commi ed p ogeni o s and
ma u e blood cells (He bein e al., 1994). HSPCs ypically
display a high nucleus- o-cy oplasm a io, wi h ew o ganelles
in he cy oplasm, while mo e di e en ia ed cells display he
opposi e (Delilie s e al., 2001). Endosomes a e pi o al in he
endocy ic pa hway and an impo an en y ou e o NPs in o
cells (Behzadi e al., 2017;Rees e al., 2019). Thus, he numbe o
endosomes inﬂuences he ex en o NP up ake and i has been
shown ha di iding HPCs ake up exogenous ma e ial much
easie han non-di iding quiescen LT-HSCs. Based on hei
TABLE 1 (Con inued) Summa y o NP sys ems a ge ing HSPCs and he BM in i o and in i o.
Ca ego y NPs Size (nm) Ta ge Pu pose Re
disease-causing mu a ion in
he β-globin gene
PLGA NPs encapsula ing PNAs and
dono DNA
156 Human HSPCs Deli e y o PNAs and DNAs o
edi he β-globin gene locus
McNee e al. (2011)
Lipid NPs encapsula ing CRISPR
(Cas9 mRNA and sgRNA)
75 Mu ine li e Inhibi ion o an i h ombin by
gene edi ing
Han e al. (2022)
Ino ganic
NPs
PEG-modiﬁed mesopo ous silica NPs 177 Mu ine ES cell-de i ed
HPCs
T acking and eal- ime
imaging o ES cell-de i ed
HPCs du ing he ea ly phases
o eng a men
Sweeney e al. (2018)
Fe umox an, magne ic polysaccha ide NPs,
ans e in, P7228 liposomes, gadopen e a e
dimeglumine liposomes SPIO and
USPIO NPs
20–40, 100–150 Human co d blood-
de i ed HSPCs
Cell labelling and MR imaging Dald up-Link e al.
(2003)
SPIO NPs co-adminis e ed wi h p o amine
sul a e
216–310 Human CD34+HSPCs Cell labelling and MR imaging England e al. (2013)
Fe umoxides–p o amine sul a e complexes Human pe iphe al
blood-de i ed HSPCs
Cell labelling and MR imaging A bab e al. (2005)
Fe2O3, Fe3O4, Sb2O3, Au, TiO2, Co and
Ag NPs
Fe3O4 (20–30),
Fe2O3 (55–65),
Sb2O3 (41–91), Au
(50–100), TiO2
(20–160), Ag
(90–210)
Human BM-de i ed
HSPCs
Cell labelling and oxici y
s udy
B egoli e al. (2009)
SPIO e umoxides NPs 120–180 Human co d blood-
de i ed HSPCs
Cell labelling and MR imaging Dald up-Link e al.
(2005)
Fluo opho e-conjuga ed dex an coa ed i on
oxide NPs
80 Human HSPCs Cell labelling and MR and
ﬂuo escen imaging
Maxwell e al. (2008)
Silica-coa ed, N-(2- aminoe hyl)-3-
aminop opyl ime hoxysilane-modiﬁed
i on NPs
60 Mu ine HSPCs Immunomagne ic cell
isola ion
Liang e al. (2009)
Fluo escen an i-CD34 an ibody-
conjuga ed- Fe3O4/Ag-NPs
30–50 Human BM-de i ed
HSPCs
Cell isola ion and de ec ion by
elec on mic oscopy
Quynh e al. (2018)
PEG-modiﬁed, guide RNA, Cp 1 (o Cas12)
endonuclease, (PEI) and single s anded
DNA empla e- unc ionalized gold NPs
64 Human HSPCs Deli e y o gene edi ing
componen s ( a ge ed HDR)
Shahbazi e al. (2019)
Na u al NPs Megaka yocy ic mic opa icles ca ying
plasmid DNA
234, 257 Human HSPCs Deli e y o nucleic acids Kao and Papou sakis,
(2018)
Plu onic/pla ele mic o esicle
nanocomplexes s abilized wi h chi osan-
algina e
467 Human HSPCs
p eloaded wi h
“nanoclouds”
Enhanced homing o
ansplan ed HSCs o he BM
Chande and
Gangenahalli, (2020b)
Baboon en elope pseudo yped “nanoblades”
used o Cas9 RNP complexes
<450 Human CD34
+
HSPCs Deli e y o he CRISPR RNP
complex o edi he WAS gene
locus
Gu ie ez-Gue e o
e al. (2021)
Hyb id NPs Polyme (PGA)-s abilized dCas9-RNP/
HDR empla e NPs
100 Human pe iphe al
blood and induced
plu ipo en s em cell
(iPS)-de i ed HSPCs
Deli e y o gene edi ing
componen s ( a ge ed HDR)
Nguyen e al. (2020)
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esis ance o in asion by ce ain bac e ia human HSCs we e long
conside ed as unable o pe o m mac opinocy osis o ecep o -
media ed phagocy osis and we e belie ed o lack he necessa y
in e naliza ion mechanisms o engul la ge amoun s o
ex acellula ma e ials (Kolb-Ma€u e e al., 2002). Howe e ,
i uses, mainly len i i us (LV, 80–100 nm), adeno i us (AdV,
90–100 nm) and adeno-associa ed i us (AAV, 25 nm), can
ansduce human HSCs, as demons a ed by ansplan a ion
expe imen s and in clinical ials (Aiu i e al., 2013;Song
e al., 2013;Geno ese e al., 2014;Sa he e al., 2015;T axle
e al., 2016;Ye e al., 2016;Kan e e al., 2017;Thompson e al.,
2018). Endocy osis is he main cellula en y ou e o i uses
lacking a i al en elope (AAV, AdV). Thus, e en hough HSCs
a e no ou s anding phagocy es, hey a e equipped o endocy ose
pa icles om hei su oundings. The size o NPs is simila o
ha o i uses, and NPs wi h di e en physical-chemical
p ope ies ha e been shown o a ge HSPCs ex i o and in
i o (Table 1). The endocy o ic ac i i y o HSPCs is also
dependen on he sou ce o CD34
+
cells. Umbilical co d blood
p ogeni o cells showed highe endocy o ic and phagocy o ic
a es compa ed o BM HSPCs (Lewin e al., 2000).
In o de o e ﬁcien ly deli e imaging eagen s o
he apeu ics o cells, en apmen ollowed by deg ada ion in
acidic compa men s o he endo/lysosomal pa hway mus be
p e en ed. Polyme ic NPs, such as hose made o poly (lac ic-
co-glycolic acid) (PLGA), ha e been shown o escape he
endosomal pa hway and ansloca e o he cy osol o
human CD34
+
HSPCs (C uz e al., 2014;C uz e al., 2021).
Di e en mechanisms, including memb ane usion, osmo ic
o mechanic up u e due o NP swelling, and memb ane
des abiliza ion by pH- esponsi e NPs ha e been p oposed
o unde lie endosomal escape and subsequen elease o
encapsula ed payload in o he cy osol (Smi h e al., 2019).
As he endosomal escape o NPs is c ucial o he e ﬁcacy o
ca go deli e y, posi i ely cha ged o pH-sensi i e unc ional
g oups can be inco po a ed in o NPs o enhance his p ocess
(Schmaljohann, 2006;Shinn e al., 2022).
1.5 In e ac ion o nanopa icles wi h
hema opoie ic s em and p ogeni o cells
Se e al g oups in es iga ed whe he polyme ic NPs a e
sui able deli e y sys ems o human HSPCs in i o (Table 1).
B üs le e al. s udied how di e en ypes o polyme ic NPs
a ec ed he unc ionali y and di e en ia ion capaci y o
human CD34
+
HSPCs (B üs le e al., 2015). Ine
polys y ene (wi hou ca boxylic g oups on he su ace) and
biodeg adable polyme ic NPs (PLGA-based) showed high
up ake a es in HSPCs wi hou inducing cy o oxici y. The
cellula NP con en was educed due o consecu i e
p oli e a ion e en s du ing lineage commi men . The
di e en ia ion po en ial o HSPCs was no a ec ed,
howe e mRNA exp ession o some lineage ma ke s was
al e ed. The signiﬁcance o his ﬁnding needs u he
in es iga ion.
De ille e al. in es iga ed he sho - e m in e ac ion and
up ake kine ics o ca boxyla ed polys y ene NPs in CD34
+
HSPCs (De ille e al., 2017). In e es ingly, in con as o
dend i ic cells, which showed inc eased NP up ake o e ime,
NP up ake in HSPCs eached a maximum wi hin 1 h and
declined a e wa ds, sugges ing an ene gy-dependen cellula
p ocess ha ac i ely con ols up ake and elease o pa icles
(De ille e al., 2017). NPs made o he na u al compound
chi osan, he only biopolyme ha is posi i ely cha ged a low
pH, ha e conside able po en ial as deli e y sys em o HSCs
based on hei abili y o deposi nega i ely cha ged molecules,
such as RNA and DNA (Cao e al., 2019). Chi osan-NPs o
di e en sizes we e explo ed as deli e y sys em o mu ine BM-
de i ed HSPCs (Zaki e al., 2015). The au ho s ound ha high
concen a ions o chi osan-NPs a ec ed cell iabili y o mouse
BM cells, in pa icula o small (200 nm) sized NPs. A low
concen a ion, medium-sized NPs educed he pe cen age o
HSCs, while in e media e and high concen a ions educed he
iabili y speciﬁcally o myeloid commi ed p ogeni o s,
indica ing size- and concen a ion-dependen cy o oxic e ec s
o chi osan NPs. NPs made o high molecula weigh chi osan
inc eased he cy o oxici y owa ds human pe iphe al blood
mononuclea cells (PBMCs) (Jesus e al., 2020). Despi e i s
biodeg adabili y, chi osan also possesses immunos imula o y
p ope ies (Han e al., 2016). Thus, u he in es iga ion is
needed o e alua e whe he chi osan-NPs a e a sui able
deli e y sys em o human HSPCs.
2 Moni o ing o hema opoie ic s em
and p ogeni o cells by nanopa icles
o nonin asi e imaging
HSC ansplan a ion ep esen s he majo cu a i e s a egy
o nume ous malignan and non-malignan hema opoie ic
diseases and is pe o med ou inely in clinical p ac ice
(Howa d e al., 2015;Labe ko and Genne y, 2018;S aal e al.,
2019). Mig a ion o ansplan ed cells o he bone ma ow niche
(‘homing’) is an impo an p e equisi e o ea men success.
Moni o ing o his p ocess helps o iden i y impai ed homing
ea ly a e ansplan a ion, allowing o in e ene o imp o e
eng a men e ﬁcacy and ansplan a ion ou come. Vi al
ec o s ha e been used o cell ma king and acking, bu
quiescen HSCs a e di ﬁcul o label by his s a egy. AdV
ec o s only ansduce cells ha unde go mi osis (Mille e al.,
1990), and LV ec o s equi e me abolic ac i i y o i al
in eg a ion (Su on e al., 1999), hus mo e e ﬁcien me hods
a e needed o allow ma king o quiescen cells. To ega d HSC
labeling and de ec ion as easible, se e al ci cums ances mus be
me . Fi s ly, he de ec ion me hod should be sensi i e enough o
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moni o labeled cells in i o. Secondly, he labeling should be
biocompa ible and p ese e he iabili y and unc ionali y o
ansplan ed HSCs. Thi dly, he cell-label associa ion should be
s able o ack HSCs o e a longe pe iod o ime. Due o a lowe
isk o label de achmen , in acellula labels deli e ed by NPs
may be p e e ed o e su ace-labeling. Labeling wi h NPs
enabled he acking o he biodis ibu ion o HSPCs using
nonin asi e biomedical imaging, such as ﬂuo escen imaging,
magne ic esonance imaging (MRI) and magne ic esonance
spec oscopy (MRS).
2.1 In i o labeling p ocedu es and
imaging o hema opoie ic s em and
p ogeni o cells
The ﬁ s s udies combining NPs and HSPCs explo ed
in i o labeling p ocedu es o p o ide ools o moni o
homing and eng a men o ansplan ed HSCs.
Supe pa amagne ic i on oxide (SPIO) and o he
in insically moni o able NPs, including gadolinium oxide-
NPs, mesopo ous silica-NPs, and PLGA-NPs encapsula ing
ﬂuo ine 19 (19
F
) as con as agen o MRI and MRS, we e
u ilized as labelling agen s o HSPCs (Table 1). Co d blood
HSPCs we e labeled wi h di e en ly sized NPs o liposomes
made o SPIO e umoxide, ul asmall SPIO (USPIO)
e umox an, ans e in-coa ed magne ic polysaccha ide,
P7228 (second-gene a ion USPIO) and gadopen e a e
dimeglumine (Dald up-Link e al., 2003). While all NPs
we e non- oxic and sui able o HSPC labeling, SPIO NPs
wi h a diame e o abou 100–150 nm we e mo e e ﬁcien ly
a ge ed o HSPCs han monoc ys alline i on oxide and
USPIO NPs, which ha e diame e s o 20–40 nm. Simila ly,
England e al. s udied he up ake o SPIO by CD34
+
HSPCs in
combina ion wi h he ans ec ion agen p o amine sul a e (a
d ug used o e e se hepa in an icoagula ion), bo h app o ed
agen s o use in pa ien s (England e al., 2013). They ound
ha he up ake o e umoxide by human HSPCs was enhanced
a e exposu e o p o amine sul a e. SPIO labeling o CD34
+
cells did no a ec cell iabili y and labeled HSPCs could be
isualized in i o by 3T MRI scanning. Simila , ano he
s a egy employed nanocomplexes o e umoxide and
p o amine sul a e o non-in asi e moni o ing o CD34
+
HSPCs by MRI (A bab e al., 2005). Labeling o HSPCs
wi h e umoxide-p o amine sul a e complexes did no
induce cellula oxici y o a ec SDF-1 induced mig a ion
and hei abili y o o m HPCs.
B egoli e al. s udied he oxici y o se en me al and me al
oxide NPs be ween 20–210 nm in size on BM CD34
+
HSPCs
(B egoli e al., 2009). Analysis o colony- o ming uni cul u es o
CD34
+
HSPCs incuba ed wi h di e en ypes o NPs showed ha
an imony oxide (Sb
2
O
3
) NPs and cobal NPs had oxic e ec s,
while he o he NPs we e non- oxic a 5, 25 and 100 μg/ml.
In e es ingly, hey ound ha Co NPs showed oxici y owa ds
e y h oid and g anulocy ic–monocy ic p ecu so s, while Sb
2
O
3
NPs we e speciﬁcally oxic o e y h oid colony de elopmen ,
sugges ing selec i e oxici y owa ds di e en HSPC
subpopula ions (B egoli e al., 2009).
In ano he app oach, Duinhouwe e al. labeled co d blood
CD34
+
HSPCs wi h PLGA-NPs con aining 19
F
(Duinhouwe
e al., 2015). NP-loaded CD34
+
HSPCs we e de ec able by MRS
in i o unde physiological condi ions. Impo an ly, he labeling
did no a ec cell iabili y and labeled CD34
+
HSPCs main ained
hei capaci y o p oli e a e and o m di e en ypes o
p ogeni o colonies in me hylcellulose assays. In a simila
app oach, HSPCs we e labeled wi h PLGA-NPs con aining
pe ﬂuo o-1,5-c own e he and imaged by MRI (Aday e al.,
2014). While he NPs did no dec ease cell iabili y, Aday
e al. demons a ed ha hese NPs modula ed he pa ac ine
ac i i y o HSPCs by dec easing he sec e ion o p o-
inﬂamma o y cy okines and a enua ing he ac i i y o oll-like
ecep o 6 and 7. Thus, hese NPs no only p o ided a con as
agen o MRI, bu also showed immunomodula o y p ope ies.
2.2 In i o acking o NP-labelled
hema opoie ic s em and p ogeni o cells
by nonin asi e imaging
In 2005, Dald up-Link e al. we e he ﬁ s o moni o human
HSPCs, loaded wi h SPIO ( e umoxide) NPs o P7228 liposomes,
a e in a enous injec ion in BALB/c mice (Dald up-Link e al.,
2005). They ound ha e umoxides we e aken up by mo e ma u e
CD34
−
,bu no byCD34
+
cells, while P7228 liposomes we e aken
up by bo h CD34
−
and CD34
+
cells. MRI analysis conﬁ med ha
i on oxide–labeled human HSPCs success ully homed o ecipien
o gans, such as he li e and spleen a 1, 4, 24 and 48 h, and he BM
a 24 and 48 h a e injec ion, wi h signal in ensi ies signiﬁcan ly
s onge han in con ols injec ed wi h pu e con as agen .
In addi ion, mul imodal PEGyla ed mesopo ous silica NPs
loaded wi h gadolinium oxide and a ﬂuo escen p obe we e
employed o MRI acking o ea ly HSPC homing in mice
(Sweeney e al., 2018). Up ake o biocompa ible mesopo ous
silica NPs by HSPCs did no a ec cell iabili y. NP-labeled
HSPCs we e acked in di e en hema opoie ic compa men s
and conﬁ med eng a men in he BM 6–9 days pos injec ion.
In e es ingly, he au ho s obse ed ha he majo i y o cells ha
had aken up mesopo ous silica NPs esembled HPCs, which
could be dis inguished in o wo mo phologically dis inc
subpopula ions wi h dis inc up ake beha io , illus a ing he
he e ogenei y o CD34
+
cell popula ions.
Maxwell e al. de eloped a mul imodal app oach o
labeling and analysis o eng a ing human HSPCs using
ﬂuo escen molecules co alen ly linked o dex an-coa ed
i on oxide NPs, allowing MR and ﬂuo escence imaging
(Maxwell e al., 2008). The ﬂuo escen label allowed o
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en ich NP+ HSPCs by ﬂuo escence-ac i a ed cell so ing p io
o ansplan a ion, as well as moni o ing o NP+ cells in i o.
Bo h quiescen and cycling HSCs we e e ﬁcien ly labeled,
wi hou inducing oxici y in i o o in i o,which
pe mi ed he dynamic acking o epopula ing HSCs
du ing he ini ial weeks a e ansplan a ion (Maxwell
e al., 2008).
3 Nanopa icles o modula e
hema opoie ic s em cell signaling
Ac i a ion o he Wingless (Wn ) pa hway is impo an o
he sel - enewal and expansion o HSPCs. Despi e some
con o e sies on he ole o Wn p o eins and Wn egula o y
ac o s in he HSC niche, ex i o cul u e o mu ine HSCs in he
FIGURE 3
HSC homing o he BM. HSC homing, as in he con ex o HSC ansplan a ion, depends on cell adhesion molecules (selec ins and in eg ins) and
ac i a ion o he SDF-1 (CXCL12)/CXCR4 axis: 1) Selec ins acili a e he ini ial e he ing/ olling o HSCs on ECs, ollowed by 2) chemokine-induced
ac i a ion o in eg ins on he HSC su ace, and 3) ﬁ m adhesion (a es ) o HSCs on he endo helium ia in eg in-in eg in ligand (LFA-1:ICAM-1, VLA-
4:VCAM-1) in e ac ions. Subsequen ly, 4) HSCs unde go diapedesis p e e en ially h ough he EC body, designa ed as anscellula
ansmig a ion, o ex a asa e ia he pa acellula ou e h ough BM ECs and each he BM niche. 5). Via in amedulla y na iga ion, HSCs mig a e o
and lodge in he endos eal and ascula niches. HCELL, hema opoie ic cell E-/L-selec in ligand; VLA-4, e y-la e an igen-4; LFA-1, lymphocy e
unc ion-associa ed an igen-1; ICAM-1, in e cellula adhesion molecule 1; VCAM-1, ascula cell adhesion molecule; SCF, s em cell ac o ; SDF-1,
s omal-de i ed ac o 1; CAR, CXCL12-abundan e icula ; PV, pe i ascula s omal; EC, endo helial cell; MSC, mesenchymal s oma cell.
F on ie s in Genome Edi ing on ie sin.o g09
C uz e al. 10.3389/ geed.2022.1030285
modula ion o he T cell ecep o and immune checkpoin
egula o s o inc ease he T cell esponse owa ds malignan
cells. In his con ex , in i o CAR-T cell induc ion media ed by
NPs encapsula ing CAR-genes and gene-edi ing ools ha e shown
p omising esul s in he ea men o leukemia. In si u p og amming
o au ologous T-cells wi h he help o NPs could a oid he sa e y
conce ns o allogeneic T cells and educe sys emic oxici ies (Xin
e al., 2022).
5.3 Nanopa icles as deli e y sys em o
gene ic he apy o hema ological
diso de s
Cu en ly, he only a ailable pe manen cu e o many
hema ological diso de s is ansplan a ion o heal hy HSCs
which ebuild he hema opoie ic sys em o myelo-abla ed
pa ien s. Bu he e is a sho age o sui able allogeneic dono s
and he ea men is linked o he isks o g a ejec ion and g a
e sus hos disease (Ca azzana e al., 2019). The u iliza ion o
gene-modiﬁed au ologous HSCs elimina es he isk o g a
e sus hos disease and nega es he necessi y o
immunosupp essi e d ugs equi ed du ing allogeneic HSC
ansplan a ion. In he pas decade, disco e y o nucleases ha
enable si e-speciﬁc genome edi ing, such as zinc-ﬁnge nucleases
(ZFN), ansc ip ion ac i a o -like e ec o nucleases (TALEN),
PNAs and clus e ed egula ly in e spaced sho palind omic
epea s (CRISPR)/CRISPR-associa ed p o ein nuclease (Cas)
(CRISPR/Cas) ha e eme ged as a ac i e ools o co ec o
amelio a e diseases o acqui ed immunodeﬁciencies, such as
hose caused by HIV, in au ologous HSCs. O e all, he
po en ial o hese ools is as , conside ing ha o e 60% o
all human disease-causing gene ic a ian s a e caused by poin
mu a ions (Rees and Liu, 2018). Among hese nucleases,
CRISPR/Cas sys ems s and ou as hey p o ide a ﬂexible,
modula , and cos -e ec i e means o edi he genome.
Gene edi ing has demons a ed o be beneﬁcial o pa ien s
wi h gene ic blood diso de s, such as sickle cell disease (SCD).
One app oach ocuses on he epai o he SCD mu a ion in he
HBB gene (SNP s334, c.20A>T, p. Glu7Val). Con incing p oo -
o -concep da a has been ob ained using homology-di ec ed
epai (HDR) o a Cas9-induced double s and b eak (DSB) a
s334 (De e e al., 2016;Uchida e al., 2021a;Uchida e al.,
2021b;La anzi e al., 2021), and base edi ing o con e he SCD
allele in o one encoding Makassa β-globin, a non-pa hogenic
a ian (Newby e al., 2021). Ano he app oach ocuses on
eac i a ion o e al hemoglobin exp ession based on educing
exp ession o BCL11A, a ansc ip ional ep esso o he e al β-
like globin genes HBG1 and HBG2 in adul e y h oid cells.
Deple ion o BCL11A in adul e y h oid cells eac i a es
HBG1/2 exp ession, which is e y beneﬁcial o SCD pa ien s.
In a ecen s udy in SCD pa ien s, CRISPR/Cas9 genome edi ing
was applied o inac i a e he e y h oid-speciﬁc enhance o he
BCL11A gene (F angoul e al., 2021), esul ing in he apeu ic
exp ession le els o γ-globin. Al e na i e app oaches ocus on
des oying he binding si e o BCL11A in he HBG1/
2 p omo e s, ei he by non-homologous end-joining o Cas9-
di ec ed DSBs, HDR o DSBs, o base edi ing, and ha e shown
p omising esul s in p e-clinical s udies (T axle e al., 2016;
Ma yn e al., 2018;Me ais e al., 2019;Wu e al., 2019;Webe
e al., 2020).
Se e al deli e y me hods a e used o pe o m CRISPR/Cas9-
media ed gene edi ing in HSPCs, including AAV (Song e al.,
2013;Sa he e al., 2015) o LV ansduc ion (T axle e al., 2016),
o elec opo a ion o ibonucleop o ein (RNP) complexes,
achie ing up o 80% e ﬁciency o gene edi ing in human
CD34
+
HSPCs (Ve hagen e al., 2022). I HDR is equi ed, he
mos e ec i e me hods ha e been elec opo a ion ollowed by
ansduc ion wi h non-in eg a ing i al ec o s (De e e al.,
2016), o concomi an elec opo a ion o RNP complexes and
chemically modiﬁed single-s anded DNA empla es (De Ra in
e al., 2017). Howe e , cu en app oaches ha e se e al
limi a ions. 1) AAV ec o s ha e a low packaging e ﬁciency
and documen ed immunogenici y, while classical i al ec o s
ca y he isk o inse ional mu agenesis (Wu e al., 2010;Yin
e al., 2014). Mo eo e long- e m Cas9 exp ession associa ed
immunogenici y can cause lysis o edi ed cells, hus u he
limi ing he use o i al ec o s (Meh a and Me kel, 2020). 2)
While success ul non- i al gene-edi ing in HSPCs using
elec opo a ion has been epo ed (Humbe e al., 2019), his
app oach emains associa ed wi h cellula oxici y (Gund y e al.,
2016;Cha leswo h e al., 2018). 3) Cu en gene edi ing
app oaches equi e ex i o cul u ing and manipula ion o
HSCs in he p esence o cy okine cock ails, which is hough
o nega i ely impac he long- e m iabili y and epopula ion
capaci y o HSCs. 4) Ex i o gene he apy o HSCs needs o be
pe o med in specialized heal hca e cen e s wi h high cos s,
es ic ing pa ien access. These limi a ions ha e inspi ed he
de elopmen o in i o deli e y sys ems, such as NPs o gene
edi ing ools, which may o e come he need o ex i o
manipula ion o pa ien HSCs, and educe o - a ge e ec s by
Cas9 ac i i y (Wilbie e al., 2019). Such de elopmen s could b ing
sa e and e ec i e gene ic he apies o all pa s o he wo ld,
including a eas o sub-Saha an A ica whe e he bu den o
diseases such as SCD and HIV a e high (Ndung’u e al., 2019;
Cannon e al., 2021).
Fo e ﬁcien gene edi ing, ools such as CRISPR need o be
adminis e ed a su ﬁcien ly high concen a ion inside a ge cells
in i o en ou e o he nucleus. DNA and RNA a e by na u e
p one o deg ada ion by se um nucleases, and possess poo
memb ane pe meabili y po en ial (Fu e al., 2014). Len i i al
ec o s a e no sui able o in i o gene edi ing, due o apid
complemen inac i a ion a e injec ion and he lack o si e-
speciﬁc a ge ing mo i s (Takeuchi e al., 1994). Se e al e iews
ha e ecen ly add essed he NP o mula ions ha ha e been
de eloped o inco po a e di e en ypes o non- i al gene edi ing
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ools (Wilbie e al., 2019;Duan e al., 2021;Naeem e al., 2021;Xu
e al., 2021). NPs p o ec hei payload and con e no el
physicochemical p ope ies o hei ca go, which enables he
e ec i e up ake o gene-edi ing componen s by he cellula
endocy osis machine y. Impo an ly, as a o emen ioned, NPs
could be equipped wi h a ge ing mo i s o HSCs in he BM
o pe iphe al blood HSCs. In pa icula , lipid NPs, polyme ic
NPs and gold NPs o e g ea po en ial as non- oxic deli e y
sys em o gene edi ing componen s (Lee e al., 2017;Finn e al.,
2018;Lee e al., 2018). Du ing he o mula ion o lipid and
polyme ic NPs, he gene edi ing componen s a e encapsula ed
in o he NP co e (C uz e al., 2021), which has he ad an age ha
he RNP complex is p o ec ed om deg ada ion du ing deli e y
o he a ge si e (Wang e al., 2016). We ha e ecen ly
demons a ed ha PLGA-NPs ep esen a sui able deli e y
sys em o co-encapsula ed RNP complexes and ﬂuo escen
p obes, and e ﬁcien ly edi ed he HBG1/2 genes in p ima y
human CD34
+
HSPCs leading o ele a ed le els o e al
hemoglobin mRNA, wi hou a ec ing hema opoie ic
p ogeni o clonogenic po en ial (C uz e al., 2021). In ano he
ecen app oach, polyme ic poly-β-amino es e (PBAE) NPs
we e used as deli e y sys em o Cas9 RNP complexes o
dis up he CD33 gene in human HSCs, a s a egy o p o ec
HSCs om an i-CD33 ea men s in acu e myeloid leukemia
pa ien s (El-Kha ag e al., 2022). Impo an ly, NP-edi ed CD34
+
and CD34
+
CD90
+
cells showed e ﬁcien long- e m eng a men
in suble hally i adia ed NSG mice and e ained mul ilineage
di e en ia ion po en ial.
FIGURE 5
Nanopa icle-media ed deli e y o gene edi ing ools o HSCs. (A–H) Schema ic illus a ion o CRISPR- and PNA-based NP-deli e y sys ems
employed o he gene ic modiﬁca ion o HSPCs. To da e, gold NPs, polyme -s abilized NPs, PLGA-NPs, lipid NPs, liposomes, i us-like pa icles
(VLPs) and EVs ha e been u ilized o he gene ic modiﬁca ion o HSPCs. (Inse ) CRISPR/Cas9 can be deli e ed as plasmid DNA, mRNA, o RNP
complex ( oge he wi h double-s anded o single-s anded DNA empla es in he case o HDR), o achie e si e-speciﬁc gene edi ing. The
di e en o ma s can be encapsula ed o su ace-deposi ed o e ﬁcien in acellula deli e y. Plasmid DNA needs o be deli e ed in o he nucleus
and be ansc ibed in o mRNA, which hen will be ansla ed in o Cas9 p o ein in he cy oplasm and be anspo ed back in o he nucleus o o m a
CRISPR RNP complex which can exe gene edi ing unc ion. Fo mRNA deli e y, he payload should be eleased in he cy osol o enable mRNA
ansla ion o p o ein. In con as , CRISPR RNP need o be deli e ed o he nucleus.
F on ie s in Genome Edi ing on ie sin.o g17
C uz e al. 10.3389/ geed.2022.1030285
In con as o polyme ic and lipid NPs, DNA, RNA and
p o ein a e commonly deposi ed on he su ace o me allic
NPs, such as gold, ia su ace modiﬁca ion chemis y o
cha ge in e ac ions (Rosi e al., 2006). In pa icula
posi i ely cha ged NPs, such as gold NPs, allow he
deposi ion o la ge amoun s o gene ic ma e ial on he NP
su ace, and he e o e ep esen an a ac i e nanoma e ial o
he deli e y o gene edi ing ools` (Laza us and Singh, 2016).
Polyme -s abilized RNP complexes can also o m NPs by
elec os a ic in e ac ions (Nguyen e al., 2020), howe e ,
simila o su ace-deposi ed gene edi ing componen s, hese
complexes a e no p o ec ed om p o eases and nucleases,
unless u he su ace unc ionaliza ion is employed. In
Figu e 5 we summa ize he cu en ly used NP-media ed
deli e y s a egies o he gene edi ing componen s CRISPR
and PNAs desc ibed in his e iew.
To es he u ili y o PLGA-NPs o he deli e y o gene
edi ing ools o HSPCs, McNee e al. encapsula ed PNAs and
dono DNA empla es con aining a desi ed sequence
modiﬁca ion in o he co e o PLGA NPs (McNee e al.,
2011). PNAs consis o nucleobases wi h a pep ide-like
backbone and enable high-a ﬁni y iplex s uc u e
o ma ion wi h DNA, igge ing DNA epai and
s imula ing DNA ecombina ion nea he PNA binding si e
(Roge s e al., 2002). Edi ing o he HBB gene locus wi h
PLGA/PNA/DNA NPs in CD34
+
HSPCs led o si e-speciﬁc
modiﬁca ions o 0.5–1% pe ea men wi hou induc ion o
cy o oxici y and p o ed o be supe io o e nucleo ec ion. This
was he ﬁ s demons a ion o biodeg adable NPs as deli e y
sys em o genome edi ing componen s. Two yea s la e ,
McNee e al. demons a ed in i o gene edi ing o he HIV
co- ecep o CCR5 ( o p e en o cu e HIV in ec ion) and HBB
gene loci in HSCs by in a enous injec ion o PLGA/PNA/
DNA NPs in a humanized mouse model, albei a low edi ing
equency (0.05% in he BM, and 0.43% in he spleen)
(McNee e al., 2013).
A ecen s udy epo ed in i o HSC gene edi ing in β-
halassemic mice using in a enously injec ed PLGA NPs,
ca ying PNAs and dono DNAs o co ec a disease-causing
mu a ion in he β-globin gene locus, in combina ion wi h SCF
gi en in ape i oneally p io o NP adminis a ion (Bahal e al.,
2016). Bahal and McNee ﬁ s epo ed he inco po a ion o
mini-PEG g oups a he γ-posi ion o some o all PNA uni s. In
i o ea men in a β- halassemic mouse model led o a gene
edi ing equency o almos 4% in o al BM cells and 6.9% in
HSCs, and imp o ed blood hemoglobin le els las ing o a leas
140 days. The au ho s ound ha SCF enhanced he PLGA/PNA/
DNA NPs-media ed gene edi ing in i o, likely a esul o
inc eased HSC mobiliza ion which may allow mo e e ﬁcien
gene ans e . In a mouse model o β- halassemia, PLGA/
PNA/DNA NPs we e also applied in a-amnio ically a
selec ed ges a ional ages wi h no impac on su i al o
pos na al g ow h. Deep sequencing e ealed co ec ion o he
disease-causing mu a ion in he HBB gene in 6% o all BM cells.
This led o a sus ained co ec ion o anemia, wi h no de ec able
o - a ge mu a ions (Riccia di e al., 2018). While PNAs lag
behind CRISPR in gene edi ing e ﬁciency, hey ha e he sa e y
ad an age o low o - a ge edi ing and no inducing double-
s anded DNA b eaks.
Ano he gene edi ing app oach o ea SCD and β-
halassemia ocusses on he in oduc ion o a speciﬁc dele ion
wi hin he HBG1/2 p omo o egion ecapi ula ing a na u al
occu ing mu a ion known as he edi a y pe sis ence o e al
hemoglobin, which is known o amelio a e disease symp oms
(Akinsheye e al., 2011). In his con ex , Shahbazi e al. de eloped
a mul ilaye PEGyla ed gold NP pla o m unc ionalized wi h
guide RNA, Cp 1 (o Cas12) endonuclease, polye hylenimine
(PEI) and single s anded DNA empla es, leading o 8.8%
HDR in CD34
+
HPSCs (Shahbazi e al., 2019). Gene edi ed
CD34
+
HPSCs eng a ed in sub-le hally i adia ed
immunodeﬁcien mice and showed s able le els o gene
edi ing o 5% in pe iphe al blood a 22 weeks pos
ansplan a ion. The au ho s ound gold NPs mo e e ﬁcien
o HDR han elec opo a ion, wi hou a ec ing HSPC
iabili y. Ins ead, hey ound a posi i e e ec o gold NPs
on he p ogeni o colony o ma ion po en ial, wi h HDR le els
ini ially dec easing a e HSC ansplan a ion be o e
e en ually s abilizing. This phenomenon was also epo ed
byo he g oups(Xu e al., 2017); he peak likely illus a es NP
up ake and gene edi ing in ma u e CD34
+
HPCs wi h limi ed
li e-span.
Recen ly, Nguyen e al. epo ed a me hod o imp o e he
e ﬁcacy o CRISPR/Cas9-based HDR in p ima y CD34
+
cells by
adding unca ed Cas9 a ge sequences a he ends o he HDR
empla e o in e ac wi h Cas9 RNPs and o shu le he empla e o
he nucleus (Nguyen e al., 2020). In addi ion, agg ega ing Cas9/
gRNA RNP complexes wi h polyglu amic acid in o NPs o 100 nm
u he imp o ed edi ing e ﬁciency o 15% in p ima y mobilized
pe iphe al blood HSPCs. Polyglu amic acid-s abilized RNP NPs
could be lyophilized, enabling upscaling o gene-modiﬁed cell
manu ac u ing o esea ch o clinical ansla ion.
Lipid NPs deli e ing Cas9 mRNA along wi h a po en
single gRNA ha e also been de eloped o he ea men o
hemophilia, a gene ic hema opoie ic diso de wi h
spon aneous bleeding caused by loss o gene unc ion in he
coagula ion pa hway (Han e al., 2022). The gRNA was
designed o a ge an i h ombin, an endogenous nega i e
egula o o h ombin gene a ion ha is encoded by he
se pin amily CC membe 1 (SERPINC1) gene. The lipid
NPs success ully deli e ed CRISPR in i o o he li e .
Th ee consecu i e doses esul ed in 50% o an i h ombin
inhibi ion and enhanced h ombosis, wi hou induc ion o
o - a ge e ec s (Han e al., 2022).
A mo e ecen echnology u ilizes “nanoblades”, consisi ing
o modiﬁed mu ine leukemia i us o HIV-de i ed i us-like
pa icles (VLP) used o RNP complexes (Gu ie ez-Gue e o
F on ie s in Genome Edi ing on ie sin.o g18
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e al., 2021). Gene edi ing wi h baboon en elope pseudo yped
nanoblades led o 40% edi ed dele ion in he Wisko -Ald ich
synd ome (WAS) gene locus in CD34
+
human HSPCs, wi hou
inducing cy o oxici y. This echnology was also combined wi h
dono -encoding AAV6 ec o s, esul ing in up o 40% o s able
exp ession casse e knock-in in o he WAS gene locus.
TABLE 2 O e iew ad an ages/disad an ages o NP-based deli e y sys em o gene edi ing componen s.
Type
o NP
Ad an age Disad an age Re
Gold NPs -Easy p epa a ion and su ace
modiﬁca ion
-No -biodeg adable Laza us and Singh (2016);Ca e y e al. (2019);Fe ei a e al. (2020);Sani
e al. (2021);Ka anagh and G een, (2022)
-High linking capaci y o gene ic
ma e ial
-Unknown long- e m oxici y
-Biocompa ible -Agg ega ion
-Tunable size and la ge su ace a ea -High cos s o la ge-scale p oduc ion
-Applicable o all ypes o CRISPR
deli e y modes
-po en ially cy o oxic
Polyme ic
NPs
-Easy p epa a ion and unable
su ace modiﬁca ion
-Unknown long- e m oxici y Bose e al. (2016);Chen e al. (2019);Duan e al. (2021);Ka anagh and
G een, (2022)
-La ge-scale p oduc ion possible -Agglome a ion
-High loading capaci y -Use o o ganic sol en s
-P o ec ion o payload om
deg ada ion
-Low oxici y
-Biodeg adable
-Low immunogenici y
-Con olled d ug elease
-Possibili y o spa io/ empo al
elease design
-Adjus able chemical and physical
p ope ies
-Excellen s abili y and long- e m
s o age
Liposomes -Easy p epa a ion -Mode a e loading capaci y Ca e y e al. (2019);Aguila -Pé ez e al. (2020);Ka anagh and G een, (2022)
-Low oxici y -Low s abili y
-Biodeg adable -Agglome a ion
-Low immunogenici y -Endosomal deg ada ion
-P o ec ion o payload om
deg ada ion
-Cos -e ﬁcien
-Can p olong d ug hal -li e
Lipid NPs -Biodeg adable -Mode a e loading capaci y o
hyd ophilic d ugs
Ghasemiyeh and Mohammadi-Samani, (2018);Ga cía-Pinel e al. (2019);
Dhiman e al. (2021)
-Biocompa ible -Payload expulsion unde s o age
condi ions
-Low oxici y -Spon aneous disin eg a ion
(polymo phic ansi ion)
-La ge-scale p oduc ion possible
-Possibili y o con olled d ug
elease
-Low immunogenici y
-Tunable su ace-modiﬁca ion
F on ie s in Genome Edi ing on ie sin.o g19
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E e y me hod o inco po a ing gene edi ing componen s
on o NP pla o ms has ad an ages and disad an ages,
and some a e be e sui ed o speciﬁc cell ypes. Table 2
shows an o e iew o he p os and cons o he mos
commonly used NP-pla o ms o deli e gene edi ing ools
o HSPCs.
6 Challenges and oppo uni ies o
nanopa icles in he ea men o
hema ological diseases
Despi e ecen de elopmen s and b eak h oughs in he
ﬁeld o NP-media ed deli e y o gene edi ing ools allowing
deploymen o CRISPR/Cas9 gene edi ing di ec ly in i o in
p ima es, including humans (Gillmo e e al., 2021;Musunu u
e al., 2021;Ro hgangl e al., 2021;Mulla d, 2022), subs an ial
obs acles emain o he deli e y o NPs o HSCs in i o. Fi s ,
se e al ex e nal and in e nal ba ie s mus be o e come ha
se e ely limi si e-speciﬁc deli e y o NPs in i o and
consequen ly a ec he apeu ic e ﬁcacy. In addi ion,
opsoniza ion and subsequen seques a ion by he
mononuclea phagocy e sys em ep esen s ano he
challenge leading o nonspeciﬁcin i o dis ibu ion and
accumula iono NPsinheal hyo gans,suchas hespleen
and he li e . Thus, NP de elope s ace he challenge o educe
non-speciﬁc accumula ion and o each he apeu ic le els a
a ge si es. PEGyla ion can signiﬁcan ly p e en
seques a ion by mononuclea phagocy es, dec ease
nonspeciﬁc dis ibu ion, unexpec ed immune esponses and
imp o e he s abili y o NPs. Howe e , he downside is he
o ma iono anaqueousphaseon heNPsu ace,which
educes he in e ac ion o NPs wi h a ge cells and hei
abili y o escape he endosomal ou e. This phenomenon is
also known as he PEG dilemma: p olonged blood ci cula ion
e sus educed cellula up ake/endosomal escape. En apmen
in endosomes/lysosomes leads o payload deg ada ion and is a
po en ial ailu e poin o NP sys ems ca ying gene edi ing
ools. Inco po a ion o pH-sensi i e compounds o clea able
chemical linke s be ween he PEG moie y and he NP su ace
can o e come en apmen in lysosomes (Schmaljohann, 2006;
Ze illo e al., 2019;Shinn e al., 2022). Upon educ ion in
pH du ing he endosomal/lysosomal ou ing, he linke s can
be clea ed o expose a posi i ely cha ged su ace o igge
endosomal escape and ansloca ion o he cy oplasm.
Modiﬁca ion o PEGyla ed NPs wi h ligands is an e ﬁcien
way o combine he ad an ages o PEG wi h cell-speciﬁc
deli e y. Mo eo e , he use o an ibodies as a ge ing
ligands has he ad an age ha cloning can be employed o
in oduce poin mu a ions in he backbone ha dec ease
an ibody-dependen cellula cy o oxici y and an ibody-
dependen cellula phagocy osis by he mononuclea
phagocy e sys em (Kang and Jung, 2019).
To a ge BM HSCs, NPs ha e o pass se e al ex e nal
ba ie s (bloods eam-EC, EC-BM, BM-LT-HSCs). This
equi es he design o e sa ile NPs ca ying mul iple
p ope ies o e he o he endo helium, pass he EC laye o
he BM niche, a ge LT-HSCs and deli e ca go e ﬁcien ly.
Scien is ecen ly epo ed e ﬁcien deli e y o CRISPR by
lipid NPs wi h BM opism. Gene edi ing o HSCs in mu ine
BM was obse ed a le els p edic ed o be cu a i e o SCD
(In ellia The apeu ics, I., 2021). Howe e , he speciﬁci y o NP
a ge ing o HSCs emains o be de e mined.
Because HSC ma ke s, including CD34, a e also p esen on
o he HPCs and ECs, i is cu en ly no possible o deli e NPs
speciﬁcally o HSCs using common HSC ma ke s. One s a egy
o inc ease binding and up ake by HSCs may be he combina ion
o NPs wi h bi alen an ibodies designed o a ge mul iple HSC
mo i s wi h lowe a ﬁni y, such ha a high-a ﬁni y in e ac ion
be ween he NP and mul iple ma ke s on HSCs would be a o ed
(Husain and Elle man, 2018). In addi ion, an imp o ed
unde s anding o HSC biology, based on s udies o pu iﬁed
HSCs, will help o de e mine which ecep o s ep esen he
mos selec i e a ge s on HSCs.
A majo conce n in he applica ion o NPs in li ing
o ganisms is sa e y and speciﬁci y. A deli e y ehicle ha can
a ge he desi ed cells wi h high-speciﬁci y will also limi o -
a ge e ec s and imp o e sa e y. I is unlikely ha a single NP
o mula ion will be uni e sally applicable o a ge exclusi ely
HSCs. Howe e , he inco po a ion o a ge ing mo i s could
g ea ly inc ease he in acellula deli e y o NPs and hei
payload o HSCs. As HSCs do no display unique cell su ace
ma ke s, and he NPs need o c oss mul iple ba ie s o each
HSCs, a a ge ing mo i , o combina ion he eo , mus be wisely
chosen o limi he complexi y o he NP o mula ion, while
inc easing BM accumula ion and up ake by HSCs. Fu u e NP
pla o ms could be de eloped o a oid p ema u e payload elease
by u ilizing bioma e ials ha espond o s imuli speciﬁcally
p esen o highly exp essed in he BM, combined wi h HSC
a ge ing mo i s o inc ease speciﬁci y owa ds HSCs.
The success o u u e gene ic he apies g ea ly depends on
ad ances in gene ic enginee ing and deli e y o he a ge cells.
Besides a ge cell deli e y issues, a cu en limi a ion in he
ansla ion o CRISPR he apies o he clinic conce ns he o -
a ge e ec s o Cas9 nucleases. High-ﬁdeli y Cas molecules wi h
educed unspeciﬁc DNA binding in combina ion wi h ansien
deli e y sys ems a e equi ed. Cas9 nickases and mu an s ha
educe non-speciﬁc DNA binding ha e been enginee ed
speciﬁcally o o e come his issue (Lino e al., 2018), which is
i al o con inued de elopmen i CRISPR/Cas9 is o ealize i s
p omise o he ea men o human diseases. E ﬁcien gene
edi ing while minimizing o - a ge e ec s is gene ally
ob ained om deli e y o he RNP complex a he han
plasmid DNA o mRNA (Kim e al., 2014). NPs allow he
ansien deli e y RNP complexes. Impo an ly, NP sys ems
can easily be adjus ed o inco po a e new a ian s o Cas
F on ie s in Genome Edi ing on ie sin.o g20
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nucleases wi h imp o ed on- a ge speciﬁci y and educed o -
a ge e ec s.
To cu e hema opoie ic diso de s, i equi es gene ic
co ec ion o LT-HSCs o elimina e o amelio a e disease
pheno ypes in hei p ogeny. Fo many inhe i ed diseases,
co ec ion o a ac ion o HSCs is su ﬁcien o e e se disease
pa hology. In SCD and b- halassemia, pos - ansplan ollow-ups
ha e shown ha mixed hema opoie ic chime ism o 10–30%
amelio a es clinical disease symp oms (Hsieh e al., 2009;
Chaudhu y e al., 2017). I NP-media ed in i o edi ing does
no achie e su ﬁcien ly high le els o chime ism a e one dose,
epe i i e dosing can easily be pe o med o inc ease chime ism
(In ellia The apeu ics, I., 2021). The si ua ion would be di e en
o malignan hema ological diso de s whe e all cells ha d i e
he disease would ha e o be a ge ed success ully. These
condi ions would he e o e be mo e di ﬁcul o ea le alone
cu e gene ically. Al e na i ely, NP-based app oaches could be
designed o achie e immune he apy o hema ological
malignancies.
NP-based diagnos ic s a egies o moni o ing o HSC g ea ly
depend on he HSC a ge ing po en ial o he deli e y sys em,
wi hou in oducing oxici y o a ec ing hei s em cell
p ope ies. Se e al nonin asi e NP-based (mul i)modali ies
ha e been de eloped o label HSPCs ex i o be o e
ansplan a ion and de ec he ini ial homing and
econs i u ion pa e ns o HSPCs wi hin a ious o gan
compa men s ele an o hema opoiesis, including he ea ly
signs o HSPC eng a men in he BM. Imp o emen in
labeling echniques and imaging p obes a e needed o long-
e m acking o HSCs. NPs a e ideally sui ed and widely used o
concomi an imaging and he apeu ic pu poses. The
inco po a ion o imaging p obes and con as agen s on gene
edi ing NP pla o ms du ing in i o gene edi ing allows in i o
moni o ing o NP dis ibu ion a he issue le el, and a he
cellula le el by ﬂow cy ome y. While HPCs p oli e a e and
dilu e NPs o e ime, i is expec ed ha NP-p obe conjuga es will
be de ec ed o longe pe iods o ime in quiescen HSCs
compa ed o HPCs, p o ided ha he imaging p ope ies o
he NPs a e su ﬁcien ly e ec i e and s able.
To build an e ﬁcien NP pla o m o he deli e y o gene
edi ing ools o HSCs, he in e ac ion be ween NPs and gene
edi ing componen s should be s ong enough o ensu e ha he
RNP complexes a e s able in he bloods eam be o e cellula
in e naliza ion. In con as , a e endosomal/lysosomal escape,
he RNP complexes should be eleased om he NPs o di use
in o he cy oplasm, and ﬁnally o ansloca e in o he nucleus.
Any p oblem a any s ep may cause he en i e deli e y p ocess o
ail. A mul ipu pose CRISPR/Cas9 deli e y sys em has s ill o
eme ge. Ra he , mul iple me hods ha e been desc ibed o deli e
CRISPR o cells. E e y me hod has i s ad an ages and
disad an ages, and some a e be e sui ed o speciﬁc cell
ypes. Ca ionic NPs (o ganic o ino ganic) s abilize he
CRISPR payload deposi ed on he NP su ace ia elec os a ic
in e ac ions, and an o ganic shell (mainly a lipid laye ) is o en
used o p o ec he RNP complexes om nucleases. Ta ge ing
ligands can be ancho ed o he shell o media e in e ac ions wi h
hos cells. On he o he hand, du ing he o mula ion o
polyme ic and lipid NPs, he RNP complexes a e encapsula ed
in o he NP co e and he e o e p o ec ed om p ema u e
deg ada ion and clea ance by he immune sys em. Polyme ic
NPs ha e he in insic ad an age ha hey display a longe shel
li e han lipid NPs. In addi ion, i is i ally impo an ha long-
e m oxici y s udies on sa e y o he NPs a e pe o med, and i
necessa y, imp o emen s in NP design a e de eloped o achie e
NPs ha a e non- oxic, non-immunogenic, and highly s able
wi h high ca go deli e y e ﬁciency. The g ea ﬂexibili y inhe en
in he use o NP-media ed gene ic he apy allows he selec ion o
he bes possible combina ion o ac o s o maximum
e ec i eness.
7 Conclusion and u u e pe spec i es
Gene edi ing echnologies, which include CRISPR/Cas
nucleases and base edi o s, hold he p omise o pe manen ly
modi y disease-causing genes in pa ien s. Despi e he exci emen
o he new b eak h oughs in gene he apy, in i o applica ion o
gene edi ing componen s is s ill in i s in ancy. Ne e heless,
se e al clinical ials o gene ic he apy ha e been comple ed
o a e unde way. This is expec ed o signiﬁcan ly inc ease o e
he nex couple o yea s and will include many ials o
hema ological diso de s.
Clea ly a numbe o challenges will ha e o be o e come.
E ﬁciency o deli e y will ha e o be imp o ed, in pa icula o
he ea men o malignan hema ological diso de s, such as
leukemias whe e mos i no all o he leukemic cells ha e o
be modiﬁed o deple ed. Speciﬁci y emains an issue because cell
su ace a ge s a e sha ed be ween di e en cell ypes and hence
o example an ibody-media ed NP deli e y will be a cause o
conce n when o he (non- a ge ed) cells a e also modiﬁed as his
may change hei unc ion, o i hey a e abundan will equi e an
un easibly high NP dose o ea men . Speciﬁci y o he edi ing
sys em i sel will also ha e o be imp o ed u he o ensu e ha
sequences o he han he in ended a ge sequences a e no
modiﬁed.
I a ge ing e ﬁciency o LT-HSCs o leukemic s em cells
would be oo low, epea ed ea men modali ies could be
conside ed o ei he inc ease he numbe o a ge ed cells o
a ge o he p ecu so cells which would ha e a sho e li e ime
han LT-HSCs. Fo example, in case o SCD ea ly e y h oid
p ogeni o cells could be co ec ed bu his would equi e
epea ed “ ea men upda es”, because such cells ha e a
limi ed li e span. In case o leukemias his may be an a enue
o keep he disease unde con ol by limi ing he numbe o
leukemic cells. Howe e , in such examples he cos pe ea men
would ha e o come down conside ably om cu en es ima es
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o gene ic he apy which ypically exceed he million-dolla
ma k pe pa ien (Leona d e al., 2022). Despi e hese challenges,
nanomedicine holds g ea p omise o he ea men o
hema ological diso de s. Recen publica ions ha e
demons a ed ha speciﬁc a ge ing can be achie ed in i o
(Wei e al., 2020;Gillmo e e al., 2021;Musunu u e al., 2021;
Ro hgangl e al., 2021;Mulla d, 2022). In ense in i o sc eens will
be necessa y o de e mine he mos op imal NP pla o m and
modiﬁca ion s a egy (Sago e al., 2018;K ohn-G imbe ghe e al.,
2020), including de elopmen o he op imal o mula ions o in
i o a ge ing o LT-HSCs.
Au ho con ibu ions
LJC and CE made subs an ial con ibu ion o he concep ,
design and w i ing o his e iew. SR, FG and SP ha e been
in ol ed in w i ing o his e iew. FG, SP, LJC and CE c i ically
e ised he manusc ip o impo an in ellec ual con en . All
au ho s ha e ead and ag eed o he published e sion o he
manusc ip .
Funding
LJC was suppo ed by p ojec g an s om he Eu opean
Commission: Ma ie Sklodowska Cu ie g an ag eemen No
777682 (CANCER), 872860 (PRISAR2), 807281 (ACORN),
852985 (SIMICA), 952520 (BIOSAFETY), 861190 (PAVE),
857894 (CAST), 859908 (NOVA-MRI) and 956477 (PIANO).
CE was suppo ed by he H2020-WIDESPREAD-2018–03
(852985-SIMICA) p ojec g an om he Eu opean
Commission. CE, LC, SP and FG we e suppo ed by he
Du ch PPS allowances made a ailable by TKI-LSH Heal h ~
Holland o he NANOCAST p ojec (EMCLSH20006 and TKI-
LSH-DT2019-LUMC: 2020–03). SR was suppo ed by he Ma ie
Sklodowska Cu ie g an ag eemen No. 857894 (CAST).
Conﬂic o in e es
The au ho s decla e ha he esea ch was conduc ed in he
absence o any comme cial o ﬁnancial ela ionships ha could
be cons ued as a po en ial conﬂic o in e es .
Publishe ’s no e
All claims exp essed in his a icle a e solely hose o he
au ho s and do no necessa ily ep esen hose o hei a ﬁlia ed
o ganiza ions, o hose o he publishe , he edi o s and he
e iewe s. Any p oduc ha may be e alua ed in his a icle, o
claim ha may be made by i s manu ac u e , is no gua an eed o
endo sed by he publishe .
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