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

Nanoparticles targeting hematopoietic stem and progenitor cells: Multimodal carriers for the treatment of hematological diseases Luis J. Cruz 1 , Somayeh Rezaei 1 , Frank Grosveld 2 , Sjaak Philipsen 2 and Christina Eich 1 * 1 Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, Leiden, Netherlands, 2 Erasmus University Medical Center, Department of Cell Biology, Rotterdam, Netherlands Modern-day hematopoietic stem cell (HSC) therapies, such as gene therapy, modify autologous HSCs prior to re-infusion into myelo-conditioned patients and hold great promise for treatment of hematological disorders. While this approach has been successful in numerous clinical trials, it relies on transplantation of ex vivo modified patient HSCs, which presents several limitations. It is a costly and time-consuming procedure, which includes only few patients so far, and ex vivo culturing negatively impacts on the viability and stem cell-properties of HSCs. If viral vectors are used, this carries the additional risk of insertional mutagenesis. A therapy delivered to HSCs in vivo, with minimal disturbance of the HSC niche, could offer great opportunities for novel treatments that aim to reverse disease symptoms for hematopoietic disorders and could bring safe, effective and affordable genetic therapies to all parts of the world. However, substantial unmet needs exist with respect to the in vivo delivery of therapeutics to HSCs. In the last decade, in particular with the development of gene editing technologies such as CRISPR/ Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the manipulation of cells and organs. By employing surface modification strategies, different types of NPs can be designed to target specific tissues and cell types in vivo. HSCs are particularly difficult to target due to the lack of unique cell surface markers that can be utilized for cell-specific delivery of therapeutics, and their shielded localization in the bone marrow (BM). Recent advances in NP technology and genetic engineering have resulted in the development of advanced nanocarriers that can deliver therapeutics and imaging agents to hematopoietic stemand progenitor cells (HSPCs) in the BM niche. In this review we provide a comprehensive overview of NP-based approaches targeting HSPCs to control and monitor HSPC activity in vitro and in vivo, and we discuss the potential of NPs for the treatment of malignant and nonmalignant hematological disorders, with a specific focus on the delivery of gene editing tools. OPEN ACCESS EDITED BY Yizong Hu, Johns Hopkins University, United States REVIEWED BY Maria Rosa Lidonnici, San Raffaele Telethon Institute for Gene Therapy, Italy Srujan Marepally, Center for Stem Cell Research, India *CORRESPONDENCE Christina Eich, [email protected] SPECIALTY SECTION This article was submitted to Genome Editing Tools and Mechanisms, a section of the journal Frontiers in Genome Editing RECEIVED 28 August 2022 ACCEPTED 10 October 2022 PUBLISHED 02 November 2022 CITATION Cruz LJ, Rezaei S, Grosveld F, Philipsen S and Eich C (2022), Nanoparticles targeting hematopoietic stem and progenitor cells: Multimodal carriers for the treatment of hematological diseases. Front. Genome Ed. 4:1030285. doi: 10.3389/fgeed.2022.1030285 COPYRIGHT © 2022 Cruz, Rezaei, Grosveld, Philipsen and Eich. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Genome Editing frontiersin.org01 TYPE Review PUBLISHED 02 November 2022 DOI 10.3389/fgeed.2022.1030285 KEYWORDS hematopoietic stem cells, nanoparticles, targeting, gene therapy, imaging, delivery 1 Introduction 1.1 Scope of this review Hematopoietic stem cells (HSCs) have the capacity to replenish all blood cell lineages during the steady-state cellular turnover of the blood system, and under stress conditions such as acute inflammation or HSC mobilization (Orkin and Zon, 2008;Sun et al., 2014;Rodriguez-Fraticelli et al., 2018). Distinct lineagecommitted hematopoietic progenitor cells (HPCs) emerge from an individual HSC through many differentiation steps and cell divisions, while HSCs are also maintained through self-renewal (Orford and Scadden, 2008;Notta et al., 2016;Velten et al., 2017; Rodriguez-Fraticelli et al., 2018). Hereditary hematological disorders (including hemophilia, blood clotting disorders, thalassemias and sickle cell disease) and acquired disorders (including myelodysplastic syndromes and malignancies such as lymphomas, leukemias and myelomas) are examples of pathologies affecting the hematopoietic system, many of which are caused by mutations (Bao et al., 2019). One way to treat these disorders is replacement of diseased HSCs by healthy allogeneic HSCs (Gyurkocza et al., 2010). HSC transplantation is one of the major medical discoveries of the 20th century and has been used for over 50 years for the treatment of leukemias and monogenic blood-related diseases (Copelan, 2006;Appelbaum, 2007). While HSC transplantations save tens of thousands of lives per year worldwide (Gratwohl et al., 2010), many patients remain deprived of this life-saving procedure due to the lack of a compatibledonororaninsufficient number of HSCs in the graft. This can lead to treatment-related morbidity and mortality (Mimeault et al., 2007). Alternatively, repair of disease-causing genes in autologous HSCs ex vivo followed by HSC transplantation or direct treatment in vivo might be the “holy grail”for malignant and non-malignant hematological diseases, provided that sufficient numbers of autologous HSCs could be corrected. In the last decade, in particular with the development of gene editing technologies such as CRISPR/Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the genetic manipulation of cells and tissues in vitro and in vivo. NPs are submicron-sized particles that can be generated from a variety of components, including polymers, lipids, metals and rare earth elements, or can be isolated as extracellular vesicles (EVs) from cells or assembled from virus capsids (Figure 1). NPs represent promising tools for monitoring and controlling HSC activity in vivo due to their capacity to protect a payload from premature degradation and mediate endosomal escape to enable nucleic acid and Cas9 translocation to the cytoplasm and nucleus (Yin et al., 2014), while circumventing efficacy and safety issues of classical viral vehicles. Moreover, recent FIGURE 1 Schematic representation of different types of nanoparticles commonly used to deliver therapeutics and imaging agents in biomedical applications. Frontiers in Genome Editing frontiersin.org02 Cruz et al. 10.3389/fgeed.2022.1030285 developments in nanotechnology have demonstrated the feasibility of site-specific delivery by smart polymers featuring spatiotemporal release kinetics (Zhuo et al., 2021), which could facilitate the manipulation of HSCs in situ while limiting off-target delivery. Several NP formulations have been approved for clinical use, mostly as a delivery vehicle for therapeutics in the field of cancer and regenerative medicine, as a delivery platform for medical imaging agents, or as a vaccine in the field of infectious diseases (Anselmo and Mitragotri, 2019). NP-based diagnostics and therapies have also received considerable attention in the field of hematological disorders, such as for the detection of circulating tumor cells by anti-CD20-coated quantum dots (Shariatifar et al., 2019), for the treatment of anemia by orally administered iron-based NPs or the manipulation of hematopoietic stemand progenitor cells (HSPCs) in the fetal and adult hematopoietic niche (Zariwala et al., 2013;Hosny et al., 2015). In this review, we provide a comprehensive overview of NPbased approaches targeting HSPCs in biomedical applications. In the first part, we focus on how HSPCs interact with NPs, taking into account the specific biology of HSCs and HPCs, and their localization in hematopoietic niches. In the second part, we review the use of NPs to control and monitor HSPC activity in vitro and in vivo. In the third part of this review, we discuss the potential of NPs for the treatment of malignant and nonmalignant hematological disorders, with a specific focus on the delivery of gene editing tools. 1.2 Nanoparticles NPs are widely accepted to have a size between 5–300 nm (although structures of up to 1,000 nm have also been reported). Based on their chemical composition, they are commonly grouped into carbon-based NPs (carbon nanotubes and fullerenes), inorganic NPs (quantum dots, metallic NPs, rare earth-material NPs) and organic NPs (lipid NPs, polymeric NPs and EVs) (Figure 1). The use of NPs as drug delivery system has many advantages over the delivery of naked drugs: 1) due to their large inner volume, NPs can be loaded with hydrophilic and hydrophobic compounds, including fluorophores, metals, peptides, proteins, nucleic acids or biomimetic molecules, which can increase the concentration of these compounds locally; 2) drug encapsulation in NPs can improve the biocompatibility and stability of conventional drugs and overcome problems of insolubility; 3) in contrast to conventional drugs, NPs present an enhanced circulation time in the blood stream; 4) NPs can be designed to be multifunctional by exerting both diagnostic and therapeutic actions; and 5) the NP surface can be functionalized with targeting moieties to permit siteand/or cell-specific payload delivery and improve the ratio of efficacy/ cytotoxicity of the encapsulated payload. This reduces adverse side effects often associated with systemically applied high doses of drugs. 1.3 Nanoparticle uptake NP uptake is influenced by three main factors: 1) The physicochemical properties of NPs, such as size, polydispersity index (measure of the heterogeneity of a sample based on size), shape, charge, surface modification and surface hydrophobicity/ hydrophilicity; 2) The physiological properties of target cells and their microenvironment (e.g., presence of cell surface proteoglycans or receptors, levels of serum proteins); and 3) experimental factors, including temperature, incubation time, osmolarity and ionic strength (He et al., 2021). After encounter with the cell membrane, NPs are taken up via the cellular endocytosis machinery by two main mechanisms, phagocytosis and pinocytosis. Phagocytosis is the preferred uptake mechanism for larger particles (>500 nm), such as pathogens, cell fragments, or NPs. Pinocytosis (including macropinocytosis, clathrin-mediated endocytosis, caveolaemediated endocytosis and clathrinand caveolin-independent endocytosis) is regarded as the dominant mechanism for the uptake of NPs of less than 500 nm (Zhao and Stenzel, 2018). Positively charged NPs are internalized rapidly via the clathrinmediated pathway, while negatively charged NPs are internalized mainly through pathways other than clathrin and caveolin (Harush-Frenkel et al., 2007). However, positively charged NPs are in general associated with higher cytotoxicity (Goodman et al., 2004;Oh et al., 2010), thus fast uptake rates are not necessarily beneficial. Current NP-based approaches targeting HSCs in biomedical applications focus around three major objectives (Figure 2). Firstly, improving labelling strategies to monitor transplanted HSCs by different imaging modalities. Secondly, the delivery of drugs and gene editing tools to modulate HSCs and the BM niche for the development of human therapeutics, and thirdly, fundamental research to develop novel tools to target and track NPs biodistribution in vivo. Examples of NPs that have been used to target HSPCs for therapy or monitoring purposes mainly belong to the group of organic and inorganic NPs (Table 1). 1.4 Hematopoietic stem cells and hematopoietic progenitor cells HSCs lack known unique cell surface markers that can be used for straightforward cell isolation. Instead, CD34 is commonly used to study HSPCs in the laboratory and for enrichment prior to BM transplantation (Sutherland et al., 1990;Baum et al., 1992). CD34 is a transmembrane Frontiers in Genome Editing frontiersin.org03 Cruz et al. 10.3389/fgeed.2022.1030285 glycoprotein expressed on HSPCs and many vascular endothelial cells (ECs). Thus, in addition to a few HSCs the CD34 + fraction includes ECs and immature and mature HPCs that can be further distinguished by additional markers (Doulatov et al., 2012). Progress has been made in characterizing human long-term repopulating HSCs (LT-HSCs) based on engraftment analysis of HSC populations as CD19 − CD34 + CD38 − CD45RACD49f+CD90 + (Thy-1) (Notta et al., 2016). Index-sorting in combination with RNA sequencing further revealed that especially the CLEC9A h iCD34 lo subset is enriched in LTrepopulating HSCs (Belluschi et al., 2018). Of note, different markers have been employed in studies in human (CD34 + CD90 + CD133+ (Wang et al., 2015;Schiroli et al., 2019; Ferrari et al., 2020) and CD34 + CD90 + (Michallet et al., 2000; Negrin et al., 2000)) versus non-human primates (CD34 + CD90 + CD45RA (Radtke et al., 2017;Humbert et al., 2019). As most studies on NPs and HSCs were conducted using CD34 + HSPCs, caution needs to be taken regarding the interpretation of NP targeting data towards HSCs. In this review, we will refer to CD34 + cells as HSPCs, unless stated otherwise. In contrast to HPCs, HSCs are primarily maintained in a quiescent (G0) state in specialized BM niches (Zhang et al., 2003). This state is accompanied by specific physiological properties, such as cell cycle arrest, reduced transcriptional and translational activity and unique energy metabolism (Passegué et al., 2005; Takubo et al., 2013;Yu et al., 2013;Ito et al., 2016) and FIGURE 2 Overview of NPs targeting HSPCs in bioimaging, fundamental research and the development of human therapeutics. Development of novel NP designs and bioconjugation strategies enables the application of multifunctional NPs for in in vivo imaging and therapy of HSPCs. Frontiers in Genome Editing frontiersin.org04 Cruz et al. 10.3389/fgeed.2022.1030285 TABLE 1 Summary of NP systems targeting HSPCs and the BM in vitro and in vivo. Category NPs Size (nm) Target Purpose Ref Polymeric NPs Polystyrene and PLLA NPs 116–131 Human HSPCs Study the influence of NPs on cell differentiation capacity and functionality Brüstle et al. (2015) Carboxylated polystyrene NPs 40 Human HSPCs Study NP loading behavior Deville et al. (2017) Chitosan NPs 200–700 Murine BM-derived HSPCs Study the influence of NP size on cell viability and functionality Zaki et al. (2015) Chitosan NPs 200 Human peripheral blood-derived HSPCs Study how deacetylation degree and molecular weight of chitosan affect cell viability and functionality Jesus et al. (2020) PLGA-NPs encapsulating fluorine 19 ( 19 F) 290 Human cord blood -derived HSPCs Cell labeling and MR imaging Duinhouwer et al. (2015) Protamine sulfate-modified PLGA-NPs encapsulating perfluoro-1,5-crown ether 210 Human HSPCs Cell labeling and MR imaging Aday et al. (2014) PLGA NPs encapsulating Wnt3a protein 178 ESCs Delivery and stabilization of Wnt3a protein Tuysuz et al. (2017) Chitosan/tripolyphosphate/fucoidan NPs encapsulating SDF-1 173–403 BM-MSCs Delivery of SDF-1 Huang and Liu, (2012) Alendronate-modified PLGA-PEG-NPs encapsulating bortezomib 150–200 Bone Drug delivery to BM Swami et al. (2014) Triblock co-polymer Poloxamer-40modified polystyrene NPs 60, 150, 250 Rabbit bone marrow (BM) Targeting the BM Porter et al. (1992) PLGA-NPs encapsulating CRISPR RNPs 300–400 Human HSPCs Delivery of CRISPR RNPs to edit the β-globin gene locus Cruz et al. (2021) Poly-β-amino ester NPs encapsulating CRISPR RNPs 200 GCSF-mobilized human CD34 + HSPCs Delivery of CRISPR RNPs to edit the CD33 gene locus El-Kharrag et al. (2022) PLGA-NPs encapsulating PNA and DNA NPs 150 HSCs In vivo gene editing of the CCR5 and β-globin gene loci McNeer et al. (2011); McNeer et al. (2013) Membrane glycan-modified carboxylated polystyrene NPs 40, 100, 200 Human HSPCs Study the influence of NP size and membrane-associated glycans on NP loading behavior Wathiong et al. (2019) Lipidbased NPs Liposomes encapsulating USPIO (P7228) 20–50 Human cord bloodderived HSPCs MR imaging of prelabeled HSPCs in the BM Daldrup-Link et al. (2005) Liposomes encapsulating Wnt3a protein 130–150 ESCs Delivery and stabilization of Wnt3a protein Tuysuz et al. (2017) Maleimide headgroup-modified liposomes and liposome-like synthetic NPs encapsulating GSK-3βinhibitor 230 Conjugated to murine HSCs Cell engineering/adjuvant delivery to improve outcome of HSC transplantation Stephan et al. (2010) PEG-lipid NPs encapsulating siRNA (“BM1”) 60–80 Murine BMECs Gene silencing in BMECs Sago et al. (2018) Lipid–PEG NPs encapsulating small siRNA (NicheEC-15″) 60–80 Murine BMECs Gene silencing in BMECs Krohn-Grimberghe et al. (2020) Alendronate-modified liposomes encapsulating SDF-1 gene 116, 123 Bone/Osteoblasts Bone targeted plasmid delivery for ectopic gene expression Chen et al. (2018) Lipid NPs encapsulating Cas9 mRNA and sgRNAs Human HSCs Gene editing of BM cells Intellia Therapeutics, I., 2021 RGD-PEGmodified liposomes encapsulating siRNA or doxorubicin 100–210 ECs SiRNA delivery to ECs Schiffelers et al. (2005) Multilamellar lipid vesicles encapsulating GSK-3βinhibitor 496 Conjugated to murine HSCs Cell engineering/adjuvant delivery to enhance proliferation kinetics of in utero transplanted HSCs Loukogeorgakis et al. (2019) PLGA NPs encapsulating γPNAs and donor DNA 200 Murine HSPCs In utero delivery of PNAs and donor DNAs to correct a Ricciardi et al. (2018) (Continued on following page) Frontiers in Genome Editing frontiersin.org05 Cruz et al. 10.3389/fgeed.2022.1030285 distinguishes HSCs from more committed progenitors and mature blood cells (Herbein et al., 1994). HSPCs typically display a high nucleus-to-cytoplasm ratio, with few organelles in the cytoplasm, while more differentiated cells display the opposite (Deliliers et al., 2001). Endosomes are pivotal in the endocytic pathway and an important entry route for NPs into cells (Behzadi et al., 2017;Rees et al., 2019). Thus, the number of endosomes influences the extent of NP uptake and it has been shown that dividing HPCs take up exogenous material much easier than non-dividing quiescent LT-HSCs. Based on their TABLE 1 (Continued) Summary of NP systems targeting HSPCs and the BM in vitro and in vivo. Category NPs Size (nm) Target Purpose Ref disease-causing mutation in the β-globin gene PLGA NPs encapsulating PNAs and donor DNA 156 Human HSPCs Delivery of PNAs and DNAs to edit the β-globin gene locus McNeer et al. (2011) Lipid NPs encapsulating CRISPR (Cas9 mRNA and sgRNA) 75 Murine liver Inhibition of antithrombin by gene editing Han et al. (2022) Inorganic NPs PEG-modified mesoporous silica NPs 177 Murine ES cell-derived HPCs Tracking and real-time imaging of ES cell-derived HPCs during the early phases of engraftment Sweeney et al. (2018) Ferumoxtran, magnetic polysaccharide NPs, transferrin, P7228 liposomes, gadopentetate dimeglumine liposomes SPIO and USPIO NPs 20–40, 100–150 Human cord bloodderived HSPCs Cell labelling and MR imaging Daldrup-Link et al. (2003) SPIO NPs co-administered with protamine sulfate 216–310 Human CD34+HSPCs Cell labelling and MR imaging England et al. (2013) Ferumoxides–protamine sulfate complexes Human peripheral blood-derived HSPCs Cell labelling and MR imaging Arbab et 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-derived HSPCs Cell labelling and toxicity study Bregoli et al. (2009) SPIO ferumoxides NPs 120–180 Human cord bloodderived HSPCs Cell labelling and MR imaging Daldrup-Link et al. (2005) Fluorophore-conjugated dextran coated iron oxide NPs 80 Human HSPCs Cell labelling and MR and fluorescent imaging Maxwell et al. (2008) Silica-coated, N-(2aminoethyl)-3aminopropyltrimethoxysilane-modified iron NPs 60 Murine HSPCs Immunomagnetic cell isolation Liang et al. (2009) Fluorescent anti-CD34 antibodyconjugatedFe3O4/Ag-NPs 30–50 Human BM-derived HSPCs Cell isolation and detection by electron microscopy Quynh et al. (2018) PEG-modified, guide RNA, Cpf1 (or Cas12) endonuclease, (PEI) and single stranded DNA template-functionalized gold NPs 64 Human HSPCs Delivery of gene editing components (targeted HDR) Shahbazi et al. (2019) Natural NPs Megakaryocytic microparticles carrying plasmid DNA 234, 257 Human HSPCs Delivery of nucleic acids Kao and Papoutsakis, (2018) Pluronic/platelet microvesicle nanocomplexes stabilized with chitosanalginate 467 Human HSPCs preloaded with “nanoclouds” Enhanced homing of transplanted HSCs to the BM Chander and Gangenahalli, (2020b) Baboon envelope pseudotyped “nanoblades” fused to Cas9 RNP complexes <450 Human CD34 + HSPCs Delivery of the CRISPR RNP complex to edit the WAS gene locus Gutierrez-Guerrero et al. (2021) Hybrid NPs Polymer (PGA)-stabilized dCas9-RNP/ HDR template NPs 100 Human peripheral blood and induced pluripotent stem cell (iPS)-derived HSPCs Delivery of gene editing components (targeted HDR) Nguyen et al. (2020) Frontiers in Genome Editing frontiersin.org06 Cruz et al. 10.3389/fgeed.2022.1030285 resistance to invasion by certain bacteria human HSCs were long considered as unable to perform macropinocytosis or receptormediated phagocytosis and were believed to lack the necessary internalization mechanisms to engulf large amounts of extracellular materials (Kolb-Ma€urer et al., 2002). However, viruses, mainly lentivirus (LV, 80–100 nm), adenovirus (AdV, 90–100 nm) and adeno-associated virus (AAV, 25 nm), can transduce human HSCs, as demonstrated by transplantation experiments and in clinical trials (Aiuti et al., 2013;Song et al., 2013;Genovese et al., 2014;Sather et al., 2015;Traxler et al., 2016;Ye et al., 2016;Kanter et al., 2017;Thompson et al., 2018). Endocytosis is the main cellular entry route for viruses lacking a viral envelope (AAV, AdV). Thus, even though HSCs are not outstanding phagocytes, they are equipped to endocytose particles from their surroundings. The size of NPs is similar to that of viruses, and NPs with different physical-chemical properties have been shown to target HSPCs ex vivo and in vivo (Table 1). The endocytotic activity of HSPCs is also dependent on the source of CD34 + cells. Umbilical cord blood progenitor cells showed higher endocytotic and phagocytotic rates compared to BM HSPCs (Lewin et al., 2000). In order to efficiently deliver imaging reagents or therapeutics to cells, entrapment followed by degradation in acidic compartments of the endo/lysosomal pathway must be prevented. Polymeric NPs, such as those made of poly (lacticco-glycolic acid) (PLGA), have been shown to escape the endosomal pathway and translocate to the cytosol of human CD34 + HSPCs (Cruz et al., 2014;Cruz et al., 2021). Different mechanisms, including membrane fusion, osmotic or mechanic rupture due to NP swelling, and membrane destabilization by pH-responsive NPs have been proposed to underlie endosomal escape and subsequent release of encapsulated payload into the cytosol (Smith et al., 2019). As the endosomal escape of NPs is crucial for the efficacy of cargo delivery, positively charged or pH-sensitive functional groups can be incorporated into NPs to enhance this process (Schmaljohann, 2006;Shinn et al., 2022). 1.5 Interaction of nanoparticles with hematopoietic stem and progenitor cells Several groups investigated whether polymeric NPs are suitable delivery systems for human HSPCs in vitro (Table 1). Brüstle et al. studied how different types of polymeric NPs affected the functionality and differentiation capacity of human CD34 + HSPCs (Brüstle et al., 2015). Inert polystyrene (without carboxylic groups on the surface) and biodegradable polymeric NPs (PLGA-based) showed high uptake rates in HSPCs without inducing cytotoxicity. The cellular NP content was reduced due to consecutive proliferation events during lineage commitment. The differentiation potential of HSPCs was not affected, however mRNA expression of some lineage markers was altered. The significance of this finding needs further investigation. Deville et al. investigated the short-term interaction and uptake kinetics of carboxylated polystyrene NPs in CD34 + HSPCs (Deville et al., 2017). Interestingly, in contrast to dendritic cells, which showed increased NP uptake over time, NP uptake in HSPCs reached a maximum within 1 h and declined afterwards, suggesting an energy-dependent cellular process that actively controls uptake and release of particles (Deville et al., 2017). NPs made of the natural compound chitosan, the only biopolymer that is positively charged at low pH, have considerable potential as delivery system for HSCs based on their ability to deposit negatively charged molecules, such as RNA and DNA (Cao et al., 2019). Chitosan-NPs of different sizes were explored as delivery system to murine BMderived HSPCs (Zaki et al., 2015). The authors found that high concentrations of chitosan-NPs affected cell viability of mouse BM cells, in particular for small (200 nm) sized NPs. At low concentration, medium-sized NPs reduced the percentage of HSCs, while intermediate and high concentrations reduced the viability specifically of myeloid committed progenitors, indicating sizeand concentration-dependent cytotoxic effects of chitosan NPs. NPs made of high molecular weight chitosan increased the cytotoxicity towards human peripheral blood mononuclear cells (PBMCs) (Jesus et al., 2020). Despite its biodegradability, chitosan also possesses immunostimulatory properties (Han et al., 2016). Thus, further investigation is needed to evaluate whether chitosan-NPs are a suitable delivery system for human HSPCs. 2 Monitoring of hematopoietic stem and progenitor cells by nanoparticles for noninvasive imaging HSC transplantation represents the major curative strategy for numerous malignant and non-malignant hematopoietic diseases and is performed routinely in clinical practice (Howard et al., 2015;Laberko and Gennery, 2018;Staal et al., 2019). Migration of transplanted cells to the bone marrow niche (‘homing’) is an important prerequisite for treatment success. Monitoring of this process helps to identify impaired homing early after transplantation, allowing to intervene to improve engraftment efficacy and transplantation outcome. Viral vectors have been used for cell marking and tracking, but quiescent HSCs are difficult to label by this strategy. AdV vectors only transduce cells that undergo mitosis (Miller et al., 1990), and LV vectors require metabolic activity for viral integration (Sutton et al., 1999), thus more efficient methods are needed to allow marking of quiescent cells. To regard HSC labeling and detection as feasible, several circumstances must be met. Firstly, the detection method should be sensitive enough to Frontiers in Genome Editing frontiersin.org07 Cruz et al. 10.3389/fgeed.2022.1030285 monitor labeled cells in vivo. Secondly, the labeling should be biocompatible and preserve the viability and functionality of transplanted HSCs. Thirdly, the cell-label association should be stable to track HSCs over a longer period of time. Due to a lower risk of label detachment, intracellular labels delivered by NPs may be preferred over surface-labeling. Labeling with NPs enabled the tracking of the biodistribution of HSPCs using noninvasive biomedical imaging, such as fluorescent imaging, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). 2.1 In vitro labeling procedures and imaging of hematopoietic stem and progenitor cells The first studies combining NPs and HSPCs explored in vitro labeling procedures to provide tools to monitor homing and engraftment of transplanted HSCs. Superparamagnetic iron oxide (SPIO) and other intrinsically monitorable NPs, including gadolinium oxideNPs, mesoporous silica-NPs, and PLGA-NPs encapsulating fluorine 19 (19 F ) as contrast agent for MRI and MRS, were utilized as labelling agents for HSPCs (Table 1). Cord blood HSPCs were labeled with differently sized NPs or liposomes made of SPIO ferumoxide, ultrasmall SPIO (USPIO) ferumoxtran, transferrin-coated magnetic polysaccharide, P7228 (second-generation USPIO) and gadopentetate dimeglumine (Daldrup-Link et al., 2003). While all NPs were non-toxic and suitable for HSPC labeling, SPIO NPs with a diameter of about 100–150 nm were more efficiently targeted to HSPCs than monocrystalline iron oxide and USPIO NPs, which have diameters of 20–40 nm. Similarly, England et al. studied the uptake of SPIO by CD34 + HSPCs in combination with the transfection agent protamine sulfate (a drug used to reverse heparin anticoagulation), both approved agents for use in patients (England et al., 2013). They found that the uptake of ferumoxide by human HSPCs was enhanced after exposure to protamine sulfate. SPIO labeling of CD34 + cells did not affect cell viability and labeled HSPCs could be visualized in vitro by 3T MRI scanning. Similar, another strategy employed nanocomplexes of ferumoxide and protamine sulfate for non-invasive monitoring of CD34 + HSPCs by MRI (Arbab et al., 2005). Labeling of HSPCs with ferumoxide-protamine sulfate complexes did not induce cellular toxicity or affect SDF-1 induced migration and their ability to form HPCs. Bregoli et al. studied the toxicity of seven metal and metal oxide NPs between 20–210 nm in size on BM CD34 + HSPCs (Bregoli et al., 2009). Analysis of colony-forming unit cultures of CD34 + HSPCs incubated with different types of NPs showed that antimony oxide (Sb 2 O 3 ) NPs and cobalt NPs had toxic effects, while the other NPs were non-toxic at 5, 25 and 100 μg/ml. Interestingly, they found that Co NPs showed toxicity towards erythroid and granulocytic–monocytic precursors, while Sb 2 O 3 NPs were specifically toxic to erythroid colony development, suggesting selective toxicity towards different HSPC subpopulations (Bregoli et al., 2009). In another approach, Duinhouwer et al. labeled cord blood CD34 + HSPCs with PLGA-NPs containing 19 F (Duinhouwer et al., 2015). NP-loaded CD34 + HSPCs were detectable by MRS in vitro under physiological conditions. Importantly, the labeling did not affect cell viability and labeled CD34 + HSPCs maintained their capacity to proliferate and form different types of progenitor colonies in methylcellulose assays. In a similar approach, HSPCs were labeled with PLGA-NPs containing perfluoro-1,5-crown ether and imaged by MRI (Aday et al., 2014). While the NPs did not decrease cell viability, Aday et al. demonstrated that these NPs modulated the paracrine activity of HSPCs by decreasing the secretion of proinflammatory cytokines and attenuating the activity of toll-like receptor 6 and 7. Thus, these NPs not only provided a contrast agent for MRI, but also showed immunomodulatory properties. 2.2 In vivo tracking of NP-labelled hematopoietic stem and progenitor cells by noninvasive imaging In 2005, Daldrup-Link et al. were the first to monitor human HSPCs, loaded with SPIO (ferumoxide) NPs or P7228 liposomes, after intravenous injection in BALB/c mice (Daldrup-Link et al., 2005). They found that ferumoxides were taken up by more mature CD34 − ,butnotbyCD34 + cells, while P7228 liposomes were taken up by both CD34 − and CD34 + cells. MRI analysis confirmed that iron oxide–labeled human HSPCs successfully homed to recipient organs, such as the liver and spleen at 1, 4, 24 and 48 h, and the BM at 24 and 48 h after injection, with signal intensities significantly stronger than in controls injected with pure contrast agent. In addition, multimodal PEGylated mesoporous silica NPs loaded with gadolinium oxide and a fluorescent probe were employed for MRI tracking of early HSPC homing in mice (Sweeney et al., 2018). Uptake of biocompatible mesoporous silica NPs by HSPCs did not affect cell viability. NP-labeled HSPCs were tracked in different hematopoietic compartments and confirmed engraftment in the BM 6–9 days post injection. Interestingly, the authors observed that the majority of cells that had taken up mesoporous silica NPs resembled HPCs, which could be distinguished into two morphologically distinct subpopulations with distinct uptake behavior, illustrating the heterogeneity of CD34 + cell populations. Maxwell et al. developed a multimodal approach for labeling and analysis of engrafting human HSPCs using fluorescent molecules covalently linked to dextran-coated iron oxide NPs, allowing MR and fluorescence imaging (Maxwell et al., 2008). The fluorescent label allowed to Frontiers in Genome Editing frontiersin.org08 Cruz et al. 10.3389/fgeed.2022.1030285 enrich NP+ HSPCs by fluorescence-activated cell sorting prior to transplantation, as well as monitoring of NP+ cells in vivo. Both quiescent and cycling HSCs were efficiently labeled, without inducing toxicity in vitro or in vivo,which permitted the dynamic tracking of repopulating HSCs during the initial weeks after transplantation (Maxwell et al., 2008). 3 Nanoparticles to modulate hematopoietic stem cell signaling Activation of the Wingless (Wnt) pathway is important for the self-renewal and expansion of HSPCs. Despite some controversies on the role of Wnt proteins and Wnt regulatory factors in the HSC niche, ex vivo culture of murine HSCs in the FIGURE 3 HSC homing to the BM. HSC homing, as in the context of HSC transplantation, depends on cell adhesion molecules (selectins and integrins) and activation of the SDF-1 (CXCL12)/CXCR4 axis: 1) Selectins facilitate the initial tethering/rolling of HSCs on ECs, followed by 2) chemokine-induced activation of integrins on the HSC surface, and 3) firm adhesion (arrest) of HSCs on the endothelium via integrin-integrin ligand (LFA-1:ICAM-1, VLA4:VCAM-1) interactions. Subsequently, 4) HSCs undergo diapedesis preferentially through the EC body, designated as transcellular transmigration, or extravasate via the paracellular route through BM ECs and reach the BM niche. 5). Via intramedullary navigation, HSCs migrate to and lodge in the endosteal and vascular niches. HCELL, hematopoietic cell E-/L-selectin ligand; VLA-4, very-late antigen-4; LFA-1, lymphocyte function-associated antigen-1; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule; SCF, stem cell factor; SDF-1, stromal-derived factor 1; CAR, CXCL12-abundant reticular; PV, perivascular stromal; EC, endothelial cell; MSC, mesenchymal stroma cell. Frontiers in Genome Editing frontiersin.org09 Cruz et al. 10.3389/fgeed.2022.1030285 modulation of the T cell receptor and immune checkpoint regulators to increase the T cell response towards malignant cells. In this context, in vivo CAR-T cell induction mediated by NPs encapsulating CAR-genes and gene-editing tools have shown promising results in the treatment of leukemia. In situ programming of autologous T-cells with the help of NPs could avoid the safety concerns of allogeneic T cells and reduce systemic toxicities (Xin et al., 2022). 5.3 Nanoparticles as delivery system of genetic therapy for hematological disorders Currently, the only available permanent cure for many hematological disorders is transplantation of healthy HSCs which rebuild the hematopoietic system of myelo-ablated patients. But there is a shortage of suitable allogeneic donors and the treatment is linked to the risks of graft rejection and graft versus host disease (Cavazzana et al., 2019). The utilization of gene-modified autologous HSCs eliminates the risk of graft versus host disease and negates the necessity for immunosuppressive drugs required during allogeneic HSC transplantation. In the past decade, discovery of nucleases that enable site-specific genome editing, such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), PNAs and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein nuclease (Cas) (CRISPR/Cas) have emerged as attractive tools to correct or ameliorate diseases or acquired immunodeficiencies, such as those caused by HIV, in autologous HSCs. Overall, the potential of these tools is vast, considering that over 60% of all human disease-causing genetic variants are caused by point mutations (Rees and Liu, 2018). Among these nucleases, CRISPR/Cas systems stand out as they provide a flexible, modular, and cost-effective means to edit the genome. Gene editing has demonstrated to be beneficial for patients with genetic blood disorders, such as sickle cell disease (SCD). One approach focuses on the repair of the SCD mutation in the HBB gene (SNP rs334, c.20A>T, p. Glu7Val). Convincing proofof-concept data has been obtained using homology-directed repair (HDR) of a Cas9-induced double strand break (DSB) at rs334 (Dever et al., 2016;Uchida et al., 2021a;Uchida et al., 2021b;Lattanzi et al., 2021), and base editing to convert the SCD allele into one encoding Makassar β-globin, a non-pathogenic variant (Newby et al., 2021). Another approach focuses on reactivation of fetal hemoglobin expression based on reducing expression of BCL11A, a transcriptional repressor of the fetal βlike globin genes HBG1 and HBG2 in adult erythroid cells. Depletion of BCL11A in adult erythroid cells reactivates HBG1/2 expression, which is very beneficial for SCD patients. In a recent study in SCD patients, CRISPR/Cas9 genome editing was applied to inactivate the erythroid-specific enhancer of the BCL11A gene (Frangoul et al., 2021), resulting in therapeutic expression levels of γ-globin. Alternative approaches focus on destroying the binding site for BCL11A in the HBG1/ 2 promoters, either by non-homologous end-joining of Cas9directed DSBs, HDR of DSBs, or base editing, and have shown promising results in pre-clinical studies (Traxler et al., 2016; Martyn et al., 2018;Metais et al., 2019;Wu et al., 2019;Weber et al., 2020). Several delivery methods are used to perform CRISPR/Cas9mediated gene editing in HSPCs, including AAV (Song et al., 2013;Sather et al., 2015) or LV transduction (Traxler et al., 2016), or electroporation of ribonucleoprotein (RNP) complexes, achieving up to 80% efficiency of gene editing in human CD34 + HSPCs (Verhagen et al., 2022). If HDR is required, the most effective methods have been electroporation followed by transduction with non-integrating viral vectors (Dever et al., 2016), or concomitant electroporation of RNP complexes and chemically modified single-stranded DNA templates (De Ravin et al., 2017). However, current approaches have several limitations. 1) AAV vectors have a low packaging efficiency and documented immunogenicity, while classical viral vectors carry the risk of insertional mutagenesis (Wu et al., 2010;Yin et al., 2014). Moreover long-term Cas9 expression associated immunogenicity can cause lysis of edited cells, thus further limiting the use of viral vectors (Mehta and Merkel, 2020). 2) While successful non-viral gene-editing in HSPCs using electroporation has been reported (Humbert et al., 2019), this approach remains associated with cellular toxicity (Gundry et al., 2016;Charlesworth et al., 2018). 3) Current gene editing approaches require ex vivo culturing and manipulation of HSCs in the presence of cytokine cocktails, which is thought to negatively impact the long-term viability and repopulation capacity of HSCs. 4) Ex vivo gene therapy of HSCs needs to be performed in specialized healthcare centers with high costs, restricting patient access. These limitations have inspired the development of in vivo delivery systems, such as NPs for gene editing tools, which may overcome the need for ex vivo manipulation of patient HSCs, and reduce off-target effects by Cas9 activity (Wilbie et al., 2019). Such developments could bring safe and effective genetic therapies to all parts of the world, including areas of sub-Saharan Africa where the burden of diseases such as SCD and HIV are high (Ndung’u et al., 2019; Cannon et al., 2021). For efficient gene editing, tools such as CRISPR need to be administered at sufficiently high concentration inside target cells in vivo en route to the nucleus. DNA and RNA are by nature prone to degradation by serum nucleases, and possess poor membrane permeability potential (Fu et al., 2014). Lentiviral vectors are not suitable for in vivo gene editing, due to rapid complement inactivation after injection and the lack of sitespecific targeting motifs (Takeuchi et al., 1994). Several reviews have recently addressed the NP formulations that have been developed to incorporate different types of non-viral gene editing Frontiers in Genome Editing frontiersin.org16 Cruz et al. 10.3389/fgeed.2022.1030285 tools (Wilbie et al., 2019;Duan et al., 2021;Naeem et al., 2021;Xu et al., 2021). NPs protect their payload and confer novel physicochemical properties to their cargo, which enables the effective uptake of gene-editing components by the cellular endocytosis machinery. Importantly, as aforementioned, NPs could be equipped with targeting motifs to HSCs in the BM or peripheral blood HSCs. In particular, lipid NPs, polymeric NPs and gold NPs offer great potential as non-toxic delivery system for gene editing components (Lee et al., 2017;Finn et al., 2018;Lee et al., 2018). During the formulation of lipid and polymeric NPs, the gene editing components are encapsulated into the NP core (Cruz et al., 2021), which has the advantage that the RNP complex is protected from degradation during delivery to the target site (Wang et al., 2016). We have recently demonstrated that PLGA-NPs represent a suitable delivery system for co-encapsulated RNP complexes and fluorescent probes, and efficiently edited the HBG1/2 genes in primary human CD34 + HSPCs leading to elevated levels of fetal hemoglobin mRNA, without affecting hematopoietic progenitor clonogenic potential (Cruz et al., 2021). In another recent approach, polymeric poly-β-amino ester (PBAE) NPs were used as delivery system for Cas9 RNP complexes to disrupt the CD33 gene in human HSCs, a strategy to protect HSCs from anti-CD33 treatments in acute myeloid leukemia patients (El-Kharrag et al., 2022). Importantly, NP-edited CD34 + and CD34 + CD90 + cells showed efficient long-term engraftment in sublethally irradiated NSG mice and retained multilineage differentiation potential. FIGURE 5 Nanoparticle-mediated delivery of gene editing tools to HSCs. (A–H) Schematic illustration of CRISPRand PNA-based NP-delivery systems employed for the genetic modification of HSPCs. To date, gold NPs, polymer-stabilized NPs, PLGA-NPs, lipid NPs, liposomes, virus-like particles (VLPs) and EVs have been utilized for the genetic modification of HSPCs. (Inset) CRISPR/Cas9 can be delivered as plasmid DNA, mRNA, or RNP complex (together with double-stranded or single-stranded DNA templates in the case of HDR), to achieve site-specific gene editing. The different formats can be encapsulated or surface-deposited for efficient intracellular delivery. Plasmid DNA needs to be delivered into the nucleus and be transcribed into mRNA, which then will be translated into Cas9 protein in the cytoplasm and be transported back into the nucleus to form a CRISPR RNP complex which can exert gene editing function. For mRNA delivery, the payload should be released in the cytosol to enable mRNA translation to protein. In contrast, CRISPR RNP need to be delivered to the nucleus. Frontiers in Genome Editing frontiersin.org17 Cruz et al. 10.3389/fgeed.2022.1030285 In contrast to polymeric and lipid NPs, DNA, RNA and protein are commonly deposited on the surface of metallic NPs, such as gold, via surface modification chemistry or charge interactions (Rosi et al., 2006). In particular positively charged NPs, such as gold NPs, allow the deposition of large amounts of genetic material on the NP surface, and therefore represent an attractive nanomaterial for the delivery of gene editing tools` (Lazarus and Singh, 2016). Polymer-stabilized RNP complexes can also form NPs by electrostatic interactions (Nguyen et al., 2020), however, similar to surface-deposited gene editing components, these complexes are not protected from proteases and nucleases, unless further surface functionalization is employed. In Figure 5 we summarize the currently used NP-mediated delivery strategies for the gene editing components CRISPR and PNAs described in this review. To test the utility of PLGA-NPs for the delivery of gene editing tools to HSPCs, McNeer et al. encapsulated PNAs and donor DNA templates containing a desired sequence modification into the core of PLGA NPs (McNeer et al., 2011). PNAs consist of nucleobases with a peptide-like backbone and enable high-affinity triplex structure formation with DNA, triggering DNA repair and stimulating DNA recombination near the PNA binding site (Rogers et al., 2002). Editing of the HBB gene locus with PLGA/PNA/DNA NPs in CD34 + HSPCs led to site-specific modifications of 0.5–1% per treatment without induction of cytotoxicity and proved to be superior over nucleofection. This was the first demonstration of biodegradable NPs as delivery system for genome editing components. Two years later, McNeer et al. demonstrated in vivo gene editing of the HIV co-receptor CCR5 (to prevent or cure HIV infection) and HBB gene loci in HSCs by intravenous injection of PLGA/PNA/ DNA NPs in a humanized mouse model, albeit at low editing frequency (0.05% in the BM, and 0.43% in the spleen) (McNeer et al., 2013). A recent study reported in vivo HSC gene editing in βthalassemic mice using intravenously injected PLGA NPs, carrying PNAs and donor DNAs to correct a disease-causing mutation in the β-globin gene locus, in combination with SCF given intraperitoneally prior to NP administration (Bahal et al., 2016). Bahal and McNeer first reported the incorporation of mini-PEG groups at the γ-position of some or all PNA units. In vivo treatment in a β-thalassemic mouse model led to a gene editing frequency of almost 4% in total BM cells and 6.9% in HSCs, and improved blood hemoglobin levels lasting for at least 140 days. The authors found that SCF enhanced the PLGA/PNA/ DNA NPs-mediated gene editing in vivo, likely a result of increased HSC mobilization which may allow more efficient gene transfer. In a mouse model of β-thalassemia, PLGA/ PNA/DNA NPs were also applied intra-amniotically at selected gestational ages with no impact on survival or postnatal growth. Deep sequencing revealed correction of the disease-causing mutation in the HBB gene in 6% of all BM cells. This led to a sustained correction of anemia, with no detectable off-target mutations (Ricciardi et al., 2018). While PNAs lag behind CRISPR in gene editing efficiency, they have the safety advantage of low off-target editing and not inducing doublestranded DNA breaks. Another gene editing approach to treat SCD and βthalassemia focusses on the introduction of a specific deletion within the HBG1/2 promotor region recapitulating a natural occurring mutation known as hereditary persistence of fetal hemoglobin, which is known to ameliorate disease symptoms (Akinsheye et al., 2011). In this context, Shahbazi et al. developed a multilayer PEGylated gold NP platform functionalized with guide RNA, Cpf1 (or Cas12) endonuclease, polyethylenimine (PEI) and single stranded DNA templates, leading to 8.8% HDR in CD34 + HPSCs (Shahbazi et al., 2019). Gene edited CD34 + HPSCs engrafted in sub-lethally irradiated immunodeficient mice and showed stable levels of gene editing of 5% in peripheral blood at 22 weeks post transplantation. The authors found gold NPs more efficient for HDR than electroporation, without affecting HSPC viability. Instead, they found a positive effect of gold NPs on the progenitor colony formation potential, with HDR levels initially decreasing after HSC transplantation before eventually stabilizing. This phenomenon was also reported byothergroups(Xu et al., 2017); the peak likely illustrates NP uptake and gene editing in mature CD34 + HPCs with limited life-span. Recently, Nguyen et al. reported a method to improve the efficacy of CRISPR/Cas9-based HDR in primary CD34 + cells by adding truncated Cas9 target sequences at the ends of the HDR template to interact with Cas9 RNPs and to shuttle the template to the nucleus (Nguyen et al., 2020). In addition, aggregating Cas9/ gRNA RNP complexes with polyglutamic acid into NPs of 100 nm further improved editing efficiency to 15% in primary mobilized peripheral blood HSPCs. Polyglutamic acid-stabilized RNP NPs could be lyophilized, enabling upscaling of gene-modified cell manufacturing for research or clinical translation. Lipid NPs delivering Cas9 mRNA along with a potent single gRNA have also been developed for the treatment of hemophilia, a genetic hematopoietic disorder with spontaneous bleeding caused by loss of gene function in the coagulation pathway (Han et al., 2022). The gRNA was designed to target antithrombin, an endogenous negative regulator of thrombin generation that is encoded by the serpin family CC member 1 (SERPINC1) gene. The lipid NPs successfully delivered CRISPR in vivo to the liver. Three consecutive doses resulted in 50% of antithrombin inhibition and enhanced thrombosis, without induction of off-target effects (Han et al., 2022). A more recent technology utilizes “nanoblades”, consisiting of modified murine leukemia virus or HIV-derived virus-like particles (VLP) fused to RNP complexes (Gutierrez-Guerrero Frontiers in Genome Editing frontiersin.org18 Cruz et al. 10.3389/fgeed.2022.1030285 et al., 2021). Gene editing with baboon envelope pseudotyped nanoblades led to 40% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus in CD34 + human HSPCs, without inducing cytotoxicity. This technology was also combined with donor-encoding rAAV6 vectors, resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. TABLE 2 Overview advantages/disadvantages of NP-based delivery system for gene editing components. Type of NP Advantage Disadvantage Ref Gold NPs -Easy preparation and surface modification -Not-biodegradable Lazarus and Singh (2016);Caffery et al. (2019);Ferreira et al. (2020);Sani et al. (2021);Kavanagh and Green, (2022) -High linking capacity for genetic material -Unknown long-term toxicity -Biocompatible -Aggregation -Tunable size and large surface area -High costs for large-scale production -Applicable for all types of CRISPR delivery modes -potentially cytotoxic Polymeric NPs -Easy preparation and tunable surface modification -Unknown long-term toxicity Bose et al. (2016);Chen et al. (2019);Duan et al. (2021);Kavanagh and Green, (2022) -Large-scale production possible -Agglomeration -High loading capacity -Use of organic solvents -Protection of payload from degradation -Low toxicity -Biodegradable -Low immunogenicity -Controlled drug release -Possibility of spatio/temporal release design -Adjustable chemical and physical properties -Excellent stability and long-term storage Liposomes -Easy preparation -Moderate loading capacity Caffery et al. (2019);Aguilar-Pérez et al. (2020);Kavanagh and Green, (2022) -Low toxicity -Low stability -Biodegradable -Agglomeration -Low immunogenicity -Endosomal degradation -Protection of payload from degradation -Cost-efficient -Can prolong drug half-life Lipid NPs -Biodegradable -Moderate loading capacity for hydrophilic drugs Ghasemiyeh and Mohammadi-Samani, (2018);García-Pinel et al. (2019); Dhiman et al. (2021) -Biocompatible -Payload expulsion under storage conditions -Low toxicity -Spontaneous disintegration (polymorphic transition) -Large-scale production possible -Possibility of controlled drug release -Low immunogenicity -Tunable surface-modification Frontiers in Genome Editing frontiersin.org19 Cruz et al. 10.3389/fgeed.2022.1030285 Every method of incorporating gene editing components onto NP platforms has advantages and disadvantages, and some are better suited for specific cell types. Table 2 shows an overview of the pros and cons of the most commonly used NP-platforms to deliver gene editing tools to HSPCs. 6 Challenges and opportunities for nanoparticles in the treatment of hematological diseases Despite recent developments and breakthroughs in the field of NP-mediated delivery of gene editing tools allowing deployment of CRISPR/Cas9 gene editing directly in vivo in primates, including humans (Gillmore et al., 2021;Musunuru et al., 2021;Rothgangl et al., 2021;Mullard, 2022), substantial obstacles remain for the delivery of NPs to HSCs in vivo. First, several external and internal barriers must be overcome that severely limit site-specific delivery of NPs in vivo and consequently affect therapeutic efficacy. In addition, opsonization and subsequent sequestration by the mononuclear phagocyte system represents another challenge leading to nonspecificin vivo distribution and accumulationofNPsinhealthyorgans,suchasthespleen and the liver. Thus, NP developers face the challenge to reduce non-specific accumulation and to reach therapeutic levels at target sites. PEGylation can significantly prevent sequestration by mononuclear phagocytes, decrease nonspecific distribution, unexpected immune responses and improve the stability of NPs. However, the downside is the formationofanaqueousphaseontheNPsurface,which reduces the interaction of NPs with target cells and their ability to escape the endosomal route. This phenomenon is also known as the PEG dilemma: prolonged blood circulation versus reduced cellular uptake/endosomal escape. Entrapment in endosomes/lysosomes leads to payload degradation and is a potential failure point of NP systems carrying gene editing tools. Incorporation of pH-sensitive compounds or cleavable chemical linkers between the PEG moiety and the NP surface can overcome entrapment in lysosomes (Schmaljohann, 2006; Zerrillo et al., 2019;Shinn et al., 2022). Upon reduction in pH during the endosomal/lysosomal routing, the linkers can be cleaved to expose a positively charged surface to trigger endosomal escape and translocation to the cytoplasm. Modification of PEGylated NPs with ligands is an efficient way to combine the advantages of PEG with cell-specific delivery. Moreover, the use of antibodies as targeting ligands has the advantage that cloning can be employed to introduce point mutations in the backbone that decrease antibody-dependent cellular cytotoxicity and antibodydependent cellular phagocytosis by the mononuclear phagocyte system (Kang and Jung, 2019). To target BM HSCs, NPs have to pass several external barriers (bloodstream-EC, EC-BM, BM-LT-HSCs). This requires the design of versatile NPs carrying multiple properties to tether to the endothelium, pass the EC layer to the BM niche, target LT-HSCs and deliver cargo efficiently. Scientist recently reported efficient delivery of CRISPR by lipid NPs with BM tropism. Gene editing of HSCs in murine BM was observed at levels predicted to be curative for SCD (Intellia Therapeutics, I., 2021). However, the specificity of NP targeting to HSCs remains to be determined. Because HSC markers, including CD34, are also present on other HPCs and ECs, it is currently not possible to deliver NPs specifically to HSCs using common HSC markers. One strategy to increase binding and uptake by HSCs may be the combination of NPs with bivalent antibodies designed to target multiple HSC motifs with lower affinity, such that a high-affinity interaction between the NP and multiple markers on HSCs would be favored (Husain and Ellerman, 2018). In addition, an improved understanding of HSC biology, based on studies of purified HSCs, will help to determine which receptors represent the most selective targets on HSCs. A major concern in the application of NPs in living organisms is safety and specificity. A delivery vehicle that can target the desired cells with high-specificity will also limit offtarget effects and improve safety. It is unlikely that a single NP formulation will be universally applicable to target exclusively HSCs. However, the incorporation of targeting motifs could greatly increase the intracellular delivery of NPs and their payload to HSCs. As HSCs do not display unique cell surface markers, and the NPs need to cross multiple barriers to reach HSCs, a targeting motif, or combination thereof, must be wisely chosen to limit the complexity of the NP formulation, while increasing BM accumulation and uptake by HSCs. Future NP platforms could be developed to avoid premature payload release by utilizing biomaterials that respond to stimuli specifically present or highly expressed in the BM, combined with HSC targeting motifs to increase specificity towards HSCs. The success of future genetic therapies greatly depends on advances in genetic engineering and delivery to the target cells. Besides target cell delivery issues, a current limitation in the translation of CRISPR therapies to the clinic concerns the offtarget effects of Cas9 nucleases. High-fidelity Cas molecules with reduced unspecific DNA binding in combination with transient delivery systems are required. Cas9 nickases and mutants that reduce non-specific DNA binding have been engineered specifically to overcome this issue (Lino et al., 2018), which is vital for continued development if CRISPR/Cas9 is to realize its promise for the treatment of human diseases. Efficient gene editing while minimizing off-target effects is generally obtained from delivery of the RNP complex rather than plasmid DNA or mRNA (Kim et al., 2014). NPs allow the transient delivery RNP complexes. Importantly, NP systems can easily be adjusted to incorporate new variants of Cas Frontiers in Genome Editing frontiersin.org20 Cruz et al. 10.3389/fgeed.2022.1030285 nucleases with improved on-target specificity and reduced offtarget effects. To cure hematopoietic disorders, it requires genetic correction of LT-HSCs to eliminate or ameliorate disease phenotypes in their progeny. For many inherited diseases, correction of a fraction of HSCs is sufficient to reverse disease pathology. In SCD and b-thalassemia, post-transplant follow-ups have shown that mixed hematopoietic chimerism of 10–30% ameliorates clinical disease symptoms (Hsieh et al., 2009; Chaudhury et al., 2017). If NP-mediated in vivo editing does not achieve sufficiently high levels of chimerism after one dose, repetitive dosing can easily be performed to increase chimerism (Intellia Therapeutics, I., 2021). The situation would be different for malignant hematological disorders where all cells that drive the disease would have to be targeted successfully. These conditions would therefore be more difficult to treat let alone cure genetically. Alternatively, NP-based approaches could be designed to achieve immune therapy of hematological malignancies. NP-based diagnostic strategies for monitoring of HSC greatly depend on the HSC targeting potential of the delivery system, without introducing toxicity or affecting their stem cell properties. Several noninvasive NP-based (multi)modalities have been developed to label HSPCs ex vivo before transplantation and detect the initial homing and reconstitution patterns of HSPCs within various organ compartments relevant to hematopoiesis, including the early signs of HSPC engraftment in the BM. Improvement in labeling techniques and imaging probes are needed for longterm tracking of HSCs. NPs are ideally suited and widely used for concomitant imaging and therapeutic purposes. The incorporation of imaging probes and contrast agents on gene editing NP platforms during in vivo gene editing allows in vivo monitoring of NP distribution at the tissue level, and at the cellular level by flow cytometry. While HPCs proliferate and dilute NPs over time, it is expected that NP-probe conjugates will be detected for longer periods of time in quiescent HSCs compared to HPCs, provided that the imaging properties of the NPs are sufficiently effective and stable. To build an efficient NP platform for the delivery of gene editing tools to HSCs, the interaction between NPs and gene editing components should be strong enough to ensure that the RNP complexes are stable in the bloodstream before cellular internalization. In contrast, after endosomal/lysosomal escape, the RNP complexes should be released from the NPs to diffuse into the cytoplasm, and finally to translocate into the nucleus. Any problem at any step may cause the entire delivery process to fail. A multipurpose CRISPR/Cas9 delivery system has still to emerge. Rather, multiple methods have been described to deliver CRISPR to cells. Every method has its advantages and disadvantages, and some are better suited for specific cell types. Cationic NPs (organic or inorganic) stabilize the CRISPR payload deposited on the NP surface via electrostatic interactions, and an organic shell (mainly a lipid layer) is often used to protect the RNP complexes from nucleases. Targeting ligands can be anchored to the shell to mediate interactions with host cells. On the other hand, during the formulation of polymeric and lipid NPs, the RNP complexes are encapsulated into the NP core and therefore protected from premature degradation and clearance by the immune system. Polymeric NPs have the intrinsic advantage that they display a longer shelf life than lipid NPs. In addition, it is vitally important that longterm toxicity studies on safety of the NPs are performed, and if necessary, improvements in NP design are developed to achieve NPs that are non-toxic, non-immunogenic, and highly stable with high cargo delivery efficiency. The great flexibility inherent in the use of NP-mediated genetic therapy allows the selection of the best possible combination of factors for maximum effectiveness. 7 Conclusion and future perspectives Gene editing technologies, which include CRISPR/Cas nucleases and base editors, hold the promise to permanently modify disease-causing genes in patients. Despite the excitement of the new breakthroughs in gene therapy, in vivo application of gene editing components is still in its infancy. Nevertheless, several clinical trials of genetic therapy have been completed or are under way. This is expected to significantly increase over the next couple of years and will include many trials for hematological disorders. Clearly a number of challenges will have to be overcome. Efficiency of delivery will have to be improved, in particular for the treatment of malignant hematological disorders, such as leukemias where most if not all of the leukemic cells have to be modified or depleted. Specificity remains an issue because cell surface targets are shared between different cell types and hence for example antibody-mediated NP delivery will be a cause of concern when other (non-targeted) cells are also modified as this may change their function, or if they are abundant will require an unfeasibly high NP dose for treatment. Specificity of the editing system itself will also have to be improved further to ensure that sequences other than the intended target sequences are not modified. If targeting efficiency of LT-HSCs or leukemic stem cells would be too low, repeated treatment modalities could be considered to either increase the number of targeted cells or target other precursor cells which would have a shorter lifetime than LT-HSCs. For example, in case of SCD early erythroid progenitor cells could be corrected but this would require repeated “treatment updates”, because such cells have a limited life span. In case of leukemias this may be an avenue to keep the disease under control by limiting the number of leukemic cells. However, in such examples the cost per treatment would have to come down considerably from current estimates Frontiers in Genome Editing frontiersin.org21 Cruz et al. 10.3389/fgeed.2022.1030285 for genetic therapy which typically exceed the million-dollar mark per patient (Leonard et al., 2022). Despite these challenges, nanomedicine holds great promise for the treatment of hematological disorders. Recent publications have demonstrated that specific targeting can be achieved in vivo (Wei et al., 2020;Gillmore et al., 2021;Musunuru et al., 2021; Rothgangl et al., 2021;Mullard, 2022). Intense in vivo screens will be necessary to determine the most optimal NP platform and modification strategy (Sago et al., 2018;Krohn-Grimberghe et al., 2020), including development of the optimal formulations for in vivo targeting of LT-HSCs. Author contributions LJC and CE made substantial contribution to the concept, design and writing of this review. SR, FG and SP have been involved in writing of this review. FG, SP, LJC and CE critically revised the manuscript for important intellectual content. All authors have read and agreed to the published version of the manuscript. Funding LJC was supported by project grants from the European Commission: Marie Sklodowska Curie grant agreement No 777682 (CANCER), 872860 (PRISAR2), 807281 (ACORN), 852985 (SIMICA), 952520 (BIOSAFETY), 861190 (PAVE), 857894 (CAST), 859908 (NOVA-MRI) and 956477 (PIANO). CE was supported by the H2020-WIDESPREAD-2018–03 (852985-SIMICA) project grant from the European Commission. CE, LC, SP and FG were supported by the Dutch PPS allowances made available by TKI-LSH Health ~ Holland for the NANOCAST project (EMCLSH20006 and TKILSH-DT2019-LUMC: 2020–03). SR was supported by the Marie Sklodowska Curie grant agreement No. 857894 (CAST). Conflict of interest The 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. 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