Delivery of an Artificial Transcription Regulator dCas9-VPR by

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Letter

Delivery of an artificial transcription regulator dCas9-VPR by extracellular vesicles for therapeutic gene activation Duško Lainš#ek, Lucija Kadunc, Mateja Man#ek-Keber, Iva Hafner-Bratkovi#, Rok Romih, and Roman Jerala ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00192 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Delivery of an artificial transcription regulator dCas9-VPR by extracellular vesicles for therapeutic gene activation

1

Duško Lainšček , Lucija Kadunc

1, 2

, Mateja Manček Keber

1, 4

1

3

, Iva Hafner Bratkovič , Rok Romih and

1, 4*

Roman Jerala

1

Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19,

Ljubljana, 1000, Slovenia. 2

Graduate school of Biomedicine, University of Ljubljana, Ljubljana, 1000, Slovenia.

3

Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, 1000,

Slovenia. 4

EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana, 1000, Slovenia.

*

Correspondence to: [email protected]; tel. no.: +38614760335; fax no.: +38614760300; National

Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.

1 ACS Paragon Plus Environment

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ABSTRACT The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of the delivery of the system into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of various molecules, including nucleic acids and proteins. We show the packaging and delivery of the CRISPR/Cas system via EVs to the target cells, combining the advantages of both technological platforms. A genome editing with designed extracellular vesicles (GEDEX) system generated by the producer cells can transfer the designed transcriptional regulator dCas9-VPR complexed with appropriate targeting gRNAs enabling activation of gene transcription. We show functional delivery in mammalian cells as well in the animals. The therapeutic efficiency of in vivo delivery of dCas9-VPR/sgRNA GEDEX is demonstrated in a mouse model of liver damage counteracted by upregulation of the endogenous hepatocyte growth factor, demonstrating the potential for therapeutic applications.

KEY WORDS: CRISPR/Cas system, extracellular vesicles, genome editing, transcription regulation



Page 4 of 25

Biologically active molecules, such as nucleic acids or proteins, can be delivered into cells via different routes and delivery strategies. Physical methods such as transfection, microinjection, electroporation and ballistic delivery are less suitable for in vivo delivery, but viral- and non-viral vectors, which can 1

encapsulate DNA, RNA or proteins, are often used for research and therapy . Although viral vectors are highly efficient, they have limitations regarding cargo size and may represent a safety risk for therapeutic applications

2,3

1,4

. Target molecules can also be delivered via virus-like particles (VLPs) ,

5,6

lipid nanoparticles (LNPs) , liposomes, polymers, a combination of osmocytosis and propanebetaine 7

, and different chemical and nonchemical conjugates, such as cell-penetrating peptides (CPP)

1,5

,

which however have limited in vivo efficiency, however. Extracellular vesicles (EVs) represent a natural mechanism for trafficking proteins, carbohydrates, 8

lipids and nucleic acids between cells . Two major groups of EVs are microvesicles (which are shed from the plasma membrane of cells) and exosomes (which are formed from multivesicular bodies). EVs have an important physiological role in the delivery of versatile cargo that can play a role in cell 8,9

signaling, tissue regeneration, homeostasis and tumorigenesis

10

. EVs have been engineered

to

deliver selected proteins and exosomes have been loaded with designed siRNAs to knock down selected genes LoxP system

11,12

. EVs can also carry miRNAs

8

which act as transcription regulators or with a Cre-

10

.

As most tissues rapidly uptake EVs

13

we reasoned that EVs may be a suitable delivery tool for

genome editing. The RNA-guided Cas9 nuclease derived from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system represents a key technological advance in molecular biology and life sciences in general and is being widely exploited for engineering almost any cell type 14

and organism . The CRISPR/Cas system can be modified to function as a designed transcription regulator by the genetic fusion of the catalytically inactive dCas9 with different transcriptional activator or repressor domains or through interaction between regulator-aptamer fusion and modified gRNAs. Due to the versatile function and use of the CRISPR/Cas system this system presents an opportunity for therapeutic genome editing. Key components of this system have been introduced into target cells mainly via transfection or electroporation of the plasmid DNA different viral vectors

15-19

, electroporation of Cas9RNP

16,17

15

or via

6

. Cas9 mRNA can also be delivered by synthetic lipid nanoparticles , induced 7

transduction by osmocytosis or by cationic, lipid-mediated delivery of Cas9:sgRNA complexes

18

. For

therapeutic delivery of Cas9 mRNA through lipid nanoparticles an additional viral vector must be used 6

to deliver single guide RNA (sgRNA) . Exosomes, which showed hypo-immunogenicity as a delivery vehicle, have been electroporated with plasmids for sgRNA and Cas9 expression

19

. Cargo loading of

exosomes via electroporation, however represents a limiting factor as electroporation has been reported to result in cargo aggregation with consequent diminished activity in the recipient cells or organism

20,21

. Exosomes have also been specifically modified such as exosome-liposome hybrid

nanoparticles, derived from the fusion of exosomes and liposomes. Different methods have been used to load exosomes with CRISPR cargo, such as incubation of hybrid exosomes with CRISPR/Cas vectors that resulted in an uptake of the CRISPR system into hybrid exosomes, enabling gene editing 22

in mesenchymal stem cells (MSC) . MSC gene editing was shown and gene intereference has been


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23

achieved, but no gene activation and in vivo potential effect were presented. In a recent publication , another improvement in microvesicles was presented, enabling increased packaging of the Cas9 protein increasing its therapeutic delivery. In that case ARMMS microvesicles (arrestin domain containing protein 1-mediated microvesicles) were used as a delivery vehicle for the CRISPR system, but again no gene transcription regulation or therapeutic in vivo effect has been shown. Here we present a GEDEX (genome editing with designed extracellular vesicles), EV-based delivery of the CRISPR/Cas system. We show that this robust system utilizes the natural endogenous biogenesis process of EV formation and loading of all components of the CRISPR/Cas system, resulting in efficient and functional delivery of programmable endonuclease and designed transcriptional regulators into recipient cells and tissues in vitro and in vivo. We reasoned that if siRNA delivered via exosomes

11,12

resulted in gene silencing in recipient

cells then the CRISPR/Cas system, which is an RNA-protein complex, might also be packaged into EVs, taken up by the recipient cells and perform genome editing in those cells. The GEDEX particles were produced autonomously by transfected HEK293 cells, based on the endogenous EVs biogenesis process of the producer cells. To ensure packaging the Cas9/gRNA into the EVs, sgRNA and S.pyogenes Cas9 nuclease (SpCas9) were overexpressed in HEK293 cells based on transient transfection with appropriate plasmids. Overexpression of desired cargo proteins to load exosomes 10,23–25

was used to ensure CRISPR packaging into GEDEX, which is commonly used method.

The

isolated EVs, shed by the HEK293 cells were characterized with standard analytical methods for EVs 12

. By demonstrating the presence of several biochemical markers of EVs and physical properties of

isolated nanoparticles, the authenticity and minimal standard experimental requirements to define 26

extracellular vesicles was demonstrated . Dynamic light scattering (DLS) analysis of the particle size confirmed the presence of particles measuring approximately 50 nm (exosomes) and 1,000 nm (microvesicles). The enrichment of the CD63 protein and the TSG101 protein, EV-characteristic tetraspanin protein and ESCRT-I sorting complex component and the absence of apoptotic bodies marker calnexin

12,26–28

biochemically confirmed the identity of the particles as EVs (Figure 1A and

Supplementary Figure S1A), also in comparison to proteins present in cell lysates of GEDEXproducing cells (Figure 1B). No calnexin protein was observed in the EVs, demonstrating the purity of the GEDEX isolation procedure. Additionally, the integrity of EVs, carrying Cas9 and the dCas9-VPR artificial transcription factor, was determined by atomic force microscopy (Supplementary Figure S1B). Cas9 was detected in exosomes and microvesicles, separated with differential ultracentrifugation (Supplementary Figure S1C). Several approaches were investigated to further increase loading of Cas9 into the GEDEX system. Producer cells were transfected with plasmids, coding for CD9, which has a role in EV biogenesis, or for neutral sphingomyelinase (NsMase-2)

9,29

, which also plays an important role in the formation of microvesicles

and exosomes through ceramide production. To explore whether the presence of the plasma membrane targeting motif on Cas9

30

could enrich EVs with the desired RNP cargo the Cas9 we took

the same approach as Wang et al. with the arrestin motif on the Cas9 protein. We designed a variant with a C-terminal CaaX box farnesylation motif of the K-Ras4B protein. However, coexpression with



Page 6 of 25

EVs biogenesis proteins did not further enrich the GEDEX system with Cas9 and the posttranslational farnesylation motif of Cas9 did not increase EV loading with the Cas9 protein (Supplementary Figure S1D), which indicates the intrinsic affinity of the Cas9/gRNA complex for packaging in EVs. The presence of SpCas9 in the GEDEX and the absence of Cas9 protein in cell supernatant after GEDEX isolation (Supplementary Figure S1E) was confirmed with western blotting (Figure 1A). Additional physical techniques to analyse the EVs features were used. Nanoparticle tracking analysis of total EVs or separate fractions of EVs determined number of the particles (Supplementary Figure S2) in isolated GEDEX. In the total EVs we can clearly see vesicles that vary in the size range distribution, confirming the presence of microvesicles and exosomes. When we measured only exosomes, the presence of a distinct peak at 140 nm demonstrated particles that correspond to the exosomes. The analysis of microvesicle fraction indeed revealed vesicles of greater size, but also vesicles of smaller size, demonstrating either microvesicles of smaller size or that also exosomes may be present in this fraction. We next compared the particle numbers to protein concentration in GEDEX, obtained with bichinonic acid assay (Supplementary Table 1) and defined that 10 μg of total GEDEX corresponds to 8

approximately 4,516 x 10 particles. With immuno-electron microscopy (Figure 1C; Supplementary Figure S3) we confirmed the specific SpCas9 cargo within the EVs. The CRISPR system component, the Cas9 protein, was detected only within the EVs that had been prepared from the HEK293 cells transfected with Cas9/gRNA-expressing vectors and not in the control EVs, which were obtained from non-transfected cells. For quantification of the amount of Cas9 in the EVs using immunodetection, recombinant Cas9 was used as a standard. Results showed that 10 μg of isolated EVs contained 100 ng of the Cas9 protein (Supplementary Figure S4 A-B). The second component of the CRISPR system, sgRNA, was quantified via qPCR in GEDEX and in the producer and recipient GEDEX cells, but only in GEDEX, carrying Cas9RNPs (Supplementary Figure S4C). By treating GEDEX with RNase A, we showed that sgRNA is present within GEDEX and not on the surface of EVs (Figure 1D). Due to recent findings that some RNAs have higher affinity for 31

the EV loading with specific RNA binding proteins, which are found in EVs , we wondered whether sgRNA may be the limiting factor for the Cas9 loading into GEDEX. Therefore, we prepared EVs, carrying the whole CRISPR/Cas system or only the Cas9 protein. With immunodetection of Cas9 and the extracellular vesicles marker, tsg-101, we confirmed that to a certain extent Cas9 loading into GEDEX is higher, when GEDEX producer cells are transfected with both components of the CRISPR system (Supplementary Figure S4 D-E) but nevertheless overexpression of Cas9 may lead to its packaging into EVs without the sgRNA. Simple overexpression probably leads to the accumulation of the Cas9 protein in the cytosol of the producer cells. Therefore, Cas9 can be packed into GEDEX, because not all Cas9 protein is imported to the cell nuclei, despite of the nuclear localization sequence (NLS)

32,33

To fully determine that all parts of the CRISPR/Cas system are truly packed within GEDEX, we performed iodixional density gradient ultracentrifugation. By comparing fractions derived from ultracentrifugation of the control GEDEX (EVs, derived from non-transfected HEK293 cells) to the


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Cas9 GEDEX fractions, we demonstrated that the Cas9 protein and sgRNA co-segregate with the CD63 protein, an extracellular vesicle marker, within the 7

th

fraction (Supplementary Figure S5), in th

th

agreement with findings, that EVs are typically found from the 6 to 8 fractions

23,26,34

.

After verification that the GEDEX system had all the components required for functional genome editing, and that the system did not contain the plasmid for Cas9 (Figure 1E) as a contaminant, the functionality of the GEDEX platform’ for the introduction of DNA double-stranded breaks (DSB) to the genomic region of the recipient HEK293 cells was tested using a GEDEX system, comprising the Cas9 nuclease and sgRNA targeting the MYD88 gene, coding for the key adapter protein in native immunity signaling

35

in human cells. GEDEX resulted in slightly lower DSB formation in comparison to

the plasmid-mediated delivery route. Indel mutations were observed in HEK293 cells that had been treated with GEDEX or were transfected with the px330 vector, expressing MYD88 sgRNA and the Cas9 protein (Figure 1F) We next reasoned that GEDEX could be also used for in vivo for gene editing thus offering a new delivery method for Cas9. We prepared DiD-labelled GEDEX, targeting Ptgs1 gene, coding for the Prostaglandin-Endoperoxide

Synthase1

that

catalyzes

the

conversion

of

arachidonate

to

prostaglandin which has been shown to have an important role in the angiogenesis and is associated 15

with some forms of cancer . By injecting GEDEX, labeled with lipophilic dye, into animals, as 36

previously demonstrated for other exosomes , we wanted to observe GEDEX trafficking in vivo. By life fluorescence measuring in the living animals (Figure 2 A-B) we determined that GEDEX is fully distributed through the organism, which was also confirmed with ex vivo organ imaging, demonstrating that DiD-labelled GEDEX can be found within all tested organs 3 h after injection, whereas the strongest signal was observed in the liver. Because the strongest signal was in the liver, we also checked the sgRNA content in liver cells with qPCR and confirmed successful uptake of GEDEX in vivo (Supplementary Figure S6). Next, we wanted to determine whether we can achieve gene editing in vivo in different organs in adult cells. In general, the CRISPR/Cas9 system for introducing DSB in adult cells often has to be delivered by viral vectors

37

or plasmids coding for CRISPR injected

38

hydrodynamically, resulting in gene editing in the liver . For genome editing in adult cells, we prepared Ptgs1-GEDEX that was injected into the tail vein of mice. T7E1 analysis revealed that gene editing occurred in all organs examined, including the heart, lung and brain (Figure 2C). For the other in vivo model for Cas9 cleavage we chose eGFP mice to knock-out the eGFP gene via GEDEX. First we tested eGFP-GEDEX on HEK293-GFP cells. We prepared microvesicles and exosomes that contained sgRNA targeting eGFP and SpCas9. We found that both microvesicles and exosomes reduced the expression of eGFP in 293/GFP cells, a result that was confirmed with confocal microscopy (Supplementary Figure S7A). As both EV fractions could be used for genome editing, the EV fractions were not separated in the experiments that followed. Next, we prepared GFP-GEDEX, EVs that contained sgRNA targeting the GFP region and SpCas9, and tested it on the target 293/GFP cells. After 48 h, the GFP fluorescence was determined by flow cytometry. We found a concentration dependent decrease in GFP fluorescence with more than 70% of all cells exhibited decreased fluorescence (Supplementary Figure S7B-C). This result was also confirmed with qPCR (Supplementary Figure S7D). Finally,

GFP-GEDEX was introduced into B6-EGFP mice, which



Page 8 of 25

39

express GFP in all tissues and cells . GFP-GEDEX was injected intraperitoneally. After 5 days, peritoneal lavage was performed, and the peritoneal cells were analyzed, with flow cytometry, for a possible decrease in eGFP. Compared to the control mice more than 50% decrease in eGFP was observed in the peritoneal cells, demonstrating that GEDEX could be used for in vivo genome editing (Supplementary Figure S7E). Different routes of administration of GEDEX resulted in a successful genome editing in the peritoneal cells. This finding demonstrates that GEDEX has a systemic and not only local use in genome editing (Supplementary Figure S7F-G). 40

The CRISPR system can also be exploited for the transcriptional regulation . By combining dCas9 with a strong composite tri-partite activator domain, such as VP64-p65-Rta (VPR), gene expression 41

can be strongly upregulated . Previously we showed that artificial transcription factors based on TALE proteins can be delivered by the use of EVs

42

. Based on our previous work and on the data that wt

Cas9 can be delivered via EVs, we examined whether GEDEX can function as a gene transcriptional activator. Therefore, the verified EVs were designed to carry a combination of sgRNA:dCas9-VPR (Figure 3A) and the integrity of dCas9-VPR EVs was determined with atomic force microscopy (Supplementary Figure S1B) Transcriptional activation by the addition of EVs was first tested with the firefly luciferase reporter. First, the plasmid-based system on transcription upregulation of firefly-luciferase reporter was tested (Supplementary Figure S8A). The detected increase in reporter activity was 160-fold in the 10 μg microvesicle-treated cells and 88-fold in the exosome-treated recipient cells in comparison to the cells that received control (ctrl) MVs or ctrl exosomes (Supplementary Figure S8B). The appropriate plasmid ratios for producing active GEDEX were also determined (Supplementary Figure S8C). The reporter expression in the total EV-treated cells increased 470-fold compared to that in the control transfection, with only the plasmids (Supplementary Figure S8A). A notable improvement was that the largest activation of the plasmid-transfected system occurred after 48 h, in contrast to the GEDEX, where high activation was observed already after 24 h, probably because

the dCas9-VPR protein

was already synthesized, bypassing the requirement for transcription/translation in a plasmid or retroviral-based delivery system. No activation of the reporter occurred upon delivery of a dCas9-VPR in the complex with random sgRNA, showing the cargo-specific function of the delivered CRISPR complexes (Figure 3B). To fully establish the presence of the artificial transcription factor, based on dCas9-VPR/sgRNA, which is responsible for gene upregulation (within the GEDEX), we pretreated the GEDEX system with proteinase K and Triton X-100. Gene upregulation of the reporter fLUC protein was achieved despite the proteinase K treatment (Supplementary Figure S8D), confirming the 23

protective role of EVs for the cargo packed within . Strong decrease of gene upregulation was observed when GEDEX was lysed using Triton X-100, demonstrating the role of membrane integrity in GEDEX. Again, the presence of sgRNA was confirmed with qPCR (Figure 3C). We further wanted to validate whether the GEDEX system could be used in animals on a systemic level to enhance gene expression levels. HEK293 cells, transfected with the firefly luciferase reporter, were introduced into mice followed by an intravenous administration of EVs, delivering the transcription factor, based on dCas9-VPR with appropriate sgRNA. Detection of the light emitted by


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upregulation of the luciferase, triggered by the systemic delivery of the GEDEX, demonstrated in vivo regulation of the expression of the reporter gene by this platform (Figure 3D-E), showing that GEDEX can be used systemically in a living organism due the system’s ability to extravasate through the vessel fenestrations and passage through the extracellular matrix, even subcutaneously

25,43

. Again,

no signal was observed when a random sgRNA accompanying dCas9-VPR artificial transcription factor was delivered. An additional in vivo experiment was performed in which the fLUC reporter was hydrodynamically injected into mice, and then, the GEDEX was administered intravenously. Bioluminescence (BLI) was observed only in the liver, confirming the specific gene upregulation (Supplementary

Figure

S9)

probably

because

the

majority

of

the

compound

delivered

44

hydrodynamically enters the liver . After upregulation of the reporter expression, we wanted to investigate whether transcriptional upregulation could be also achieved for endogenous genes, enabling the possible use of this delivery system for a different range of applications. Therefore, GEDEX was prepared comprising dCas9-VPR 41

and sgRNA targeting the promotor region of the human and mouse Actc1 gene

(around 200 bp

upstream of the transcription start site (TSS)). By treating HEK293 cells or mouse Neuro-2A cells with EVs, carrying the dCas9-VPR and sgRNA against the human or mouse Actc1 gene, we observed a seven-fold increase in the transcript of ACTC1 in human cells and a five-fold increase in mouse cells relative to the reference gene expression showing that GEDEX can be used for transcriptional upregulation on the endogenous level in mammalian cells (Figure 4). EVs also have an important potential for tissue regeneration

45

. Based on the ability of GEDEX to

upregulate endogenous genes and to function in vivo, as demonstrated above, we set out to investigate whther GEDEX could be used for a therapeutic application. As the proof of principle of the therapeutic application, we selected upregulation of mouse Hgf (Hepatocyte Growth Factor) gene to increase HGF production, which has an important role in liver regeneration

46

. First, five candidate

sgRNAs targeting the Hgf promoter region approximately 200 bp upstream from the TSS were tested using NIH-3T3 cells. Unexpectedly, ELISA did not demonstrate any increase at the protein level with any upstream located sgRNA (Figure 5A). Gene upregulation might also be achieved by targeting the first intron or exon

47

, therefore five

additional target sgRNAs in the first exon or region downstream (up to 500 bp downstream of the TSS) were tested. In this case, gRNAs targeting the first exon or downstream genome region efficiently upregulated the mouse Hgf gene (Figure 5B), where gRNA8 performed best. Next, GEDEX comprising dCas9 and sgRNA8 was prepared to deliver GEDEX into the mouse liver.. First, we tested the possible pathological effect of the administration of GEDEX to the animal liver. By measuring a liver enzyme profile we established that hydrodynamic delivery of GEDEX did not influence the state of the liver after 4 days (Supplementary Figure S10). Therefore, we proceeded with the in vivo determination of the therapeutic effect of the GEDEX system. Injection of GEDEX targeting Hgf successfully enhanced mouse Hgf transcription (Supplementary Figure S11) in an alphanaphthylisothiocyanate (ANIT) mice liver damage model. This s a model for human drug-induced hepatotoxicity resulting from the destruction of hepatocytes or damage to the biliary liver system,



ending in cholestasis

Page 10 of 25

48,49

. Mice were given a single oral dose of ANIT, followed by hydrodynamic

delivery of GEDEX for upregulation of Hgf or control EVs. After two days, mice were sacrificed and blood and liver samples were collected. The analysis of mouse HGF in liver lysates demonstrated that GEDEX statistically significantly increased HGF in the treated animals (Figure 5C), which resulted in a therapeutic effect on liver regeneration. In mouse sera, specific liver markers were determined. In the control animals treated with ANIT and control EVs all liver biochemical markers were drastically increased. The liver damage gold standard, the ALT enzyme

48

, was approximately five-fold higher in

animals treated with ANIT than in the animals treated with ANIT and Hgf-EVs,confirming the therapeutic effect of upregulation of the Hgf gene. A similar difference was observed for the bile acids, total bilirubin and cholesterol: All liver damage markers were higher in the non-treated animals (Figure 5D). The same conclusion regarding GEDEX treatment ot the ANIT mouse model can be drawn when GEDEX was administered with a simple intravenous injection (Supplementary Figure S12). When GEDEX was injected intravenously and not hydrodynamically as stated above, we observed lower HGF increase. This confirms that hydrodynamic delivery of injected compounds leads indeed to higher accumulation in liver, thus increasing the GEDEX load in the liver and the therapeutic effect. The histological analysis also corroborated the liver marker measurements and revealed that the livers of the animals that had been administered with Hgf EVs showed significantly less liver tissue damage. In the animals that had been administered control EVs, we observed far greater tissue cell infiltration, hyperemia and the existence of necrotic areas, based on the loss of cell nuclei visualization. In animals that were given the Hgf-GEDEX we observed some cell infiltration, but far less than in the animals without Hgf upregulation, and there were no signs of necrosis development. Their liver exhibited morphology similar to the animals that had been treated only with saline solution (Figure 5E). Efficient delivery of the CRISPR/Cas9 system remains an important bottleneck particularly for in vivo therapeutic applications. We demonstrated the functional delivery of the CRISPR system in mammalian cells. Transcriptional activation was also demonstrated in animals, which, thus far, has been limited mainly to virus-based systems and had never been shown with the use of extracellular vesicles. Although protein delivery with induced transduction by osmocytosis demonstrated efficient 50

delivery of the Cas9 protein to cells in the culture , this technique is not applicable for in vivo use. The main advantages of GEDEX are facile preparation, efficiency in different cell types, in vivo efficiency and lack of toxicity, because the generated EVs closely mimic natural systems. A particularly important implementation was the use of GEDEX to upregulate endogenous gene transcription, which could be highly relevant for therapeutic applications, as essentially all genes or their combinations may be targeted. The decreased time delay for the onset of the activity is particularly relevant for potential therapeutic application through transcriptional activation or repression, demonstrated by the GEDEX targeting of Hgf. The ANIT-induced liver damage model has been previously successfully treated only with the delivery of the HGF protein

46,48

. Endogenous upregulation of Hgf has to our knowledge never

been reported by any other technique and may be applied in many liver-related diseases, such as acute liver failure, liver cirrhosis, fibrosis etc. In the case of Hgf the optimal gRNA position was


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determined within the first exon rather than upstream from the TSS, which should be remembered for gRNA screening of other targets. We conclude that the potentials for extracellular vesicles and CRISPR/Cas combined into a technological platform provides an exciting opportunity for medical therapy as well as a research tool. Additional improvements can clearly be envisaged, such as extending GEDEX by introducing targeting motifs on Cas9

23

or into EVs,as demonstrated for siRNA loaded EVs

11

or employing different 22

strategies to increase the amount of produced and isolated EVs or increasing the desired cargo load .

METHODS Cell cultures The human embryonic kidney (HEK) 293 and mouse Neuro-2a cells were purchased from American Type Culture Collection (ATCC). The 293/GFP cell line, which stably expresses GFP, was purchased from Cell Biolabs. Cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% (v/v) heat-inactivated FBS (Invitrogen Life Technologies) at 37 °C in 5% CO2. Reagents The following primary antibodies were used for western blotting or electron microscopy: rabbit antiCD63 (diluted 1:1,000; sc-15363) from Santa Cruz, mouse anti-CRISPR-Cas9 antibody (diluted 1:500; ab191468) from Abcam, mouse anti-Tsg101 from Santa Cruz (diluted 1:1,000; sc-7964), rabbit anticalnexin (diluted 1:500; ab22595) from Abcam and rabbit anti-Alpha/Beta tubulin antibody (diluted 1:1,000; 2148) purchased from Cell Signaling Technology. Secondary antibodies used for western blotting were goat polyclonal anti-rabbit IgG (diluted 1:4,000; ab6721) from Abcam and goat antimouse IgG-HRP (diluted 1:4,000; sc-2005) from Santa Cruz. Secondary antibodies used for electron microscopy were goat anti-mouse IgG conjugated with 18 nm gold from Abcam (ab39614). Plasmids Target sites for genome editing were determined by using CRISPR Design Tool from MIT. sgRNAs were cloned into BbsI site of pX330 plasmid (Addgene plasmid 42230) with ligation of annealed primers for sgRNA or were introduced via PCR into the plasmid pgRNA-humanized (Addgene plasmid 44248). dCas9 was obtained from pHR-SFFVdCas9-BFP-KRAB (Addgene plasmid 46911). The VPR activator sequence was adapted from the Addgene plasmid 63801 and synthesized by Genewiz and then cloned with Gibson assembly method into pCMV-dCas9-VPR vector. For the reporter firefly luciferase vector all sgRNA target sites were designed as seven repeats that were separated by hypervariable linkers and synthesized by Genewiz and cloned into 7sgRNA[B]-min-fLuc reporter vector. Reporter gene for firefly luciferase was PCR amplified from commercial plasmid pGL4.16 (Promega). The SV40 large T-antigen nuclear localization sequence (NLS), the hexa-histidine tag, farnesyl motif and the minimal promoter were introduced into the constructs with PCR. Renilla luciferase (phRL-TK, Promega) was used as a transfection control.



Page 12 of 25

Preparation of the GEDEX system (extracellular vesicle isolation) 6

HEK293 cells were seeded in a 10 cm tissue culture dish (TPP) at a density of 4 × 10 cells/dish in DMEM supplemented with 10% (v/v) heat-inactivated exosome-depleted FBS (SystemBio) at 37 °C in –

5% CO2. At 80 90% confluency, the cells were transfected with a mixture of DNA and PEI (12 μl/1 μg DNA). The total DNA concentration per tissue dish was 20 μg. To prepare the control (ctrl) GEDEX or EVs (EVs without Cas9 RNP), the cells were not transfected; the medium was just removed. After 24 h, the medium, containing extracellular vesicles, was collected and centrifuged at 3,000 ×g for 15 min to remove cellular debris. Isolation of the extracellular vesicles was performed with ultracentrifugation. Cell supernatants were put into appropriate centrifuge tubes and then centrifuged at 100,000 ×g for 1 h (Beckman Coulter Ultracentrifuge). The supernatant was removed, and the EV pellet was washed with cold PBS (Invitrogen Life Technologies) and then again centrifuged at 100,000 ×g for 1 h at 4 °C. For different EV fractions, after the cellular debris was removed, the cell supernatant was first centrifuged at 10,000 ×g for 40 min at 4 °C for microvesicles, and then the supernatant was again centrifuged for 1 h at 100,000 ×g at 4 °C to acquire exosomes. The EV concentration was determined with BCA using the QuantiPRO

TM

BCA Assay kit (Sigma Aldrich).

Luciferase activity assay 4

HEK293 cells were seeded in White 96-well plates (Corning) at 2 x 10 cells/well. After 24 h the cells were transfected with a mixture of DNA and PEI. Total amount of DNA per well was 200 ng. To determine the luciferase activity of plasmids coding dCas9-VPR and appropriate sgRNA, cells were transfected with pCMV-dCas9-VPR, mU6_sgRNA[B] in different ratio, 7sgRNA [B]-min-fLuc reporter vector (50 or 100 ng DNA/well) and phRL-TK (5 ng DNA/well). In order to obtain luciferase activity of GEDEX, carrying dCas9-VPR and sgRNAB, the cells were transfected with 7sgRNA [B]-min-fLuc reporter vector (50 ng DNA/well), phRL-TK (5 ng DNA/well) and up to 200 ng of DNA with empty vector pcDNA3.1 (Invitrogen). Where stated, EVs were treated with proteinase K (100 μg/ml; Qiagen) for 1 hour at 37 °C or permeabilized with the addition of 0,1% Triton X-100 (Sigma Aldrich). Proteinase K was heat inactivated for 15 minutes at 70 °C. After the treatment, EVs were used to stimulate the transfected cells. After 24 h cell medium was replaced and the cells were treated with different concentrations of GEDEX. Twenty-four hours post EV treatment or 48 h in case of determining plasmid luciferase activity; the cells were harvested and lysed in Passive Lysis Buffer (Promega). The expression of the luciferase reporter genes was analyzed using Dual Glo Luciferase Assay System reagents (Promega) and the Orion luminometer plate reader (Berthold Detection Systems). Relative luciferase activity was calculated by normalizing each sample’s firefly luciferase activity with the constitutive Renilla luciferase activity determined within the same sample. Semi-quantitative PCR Hgf mRNA was isolated by using the High Pure RNA Isolation Kit (Roche) according to the manufacturer’s protocol. For the detection of sgRNA in extracellular vesicles, different EV fractions from the DGUC and the samples, we used the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Where stated, the EVs were first treated with RNase A (ThermoFisher Scientific) at 37 °C for 15 min, and then RNA was isolated. One microgram of mRNA was transcribed


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with the High Capacity cDNA Transcription Kit (AB Applied Biosystems). qPCR was performed using R

the LightCycler 480 SYBR Green I Master mix (Roche) on the LightCycler 480 instrument (Roche). T7E1 assay Genomic DNA from EVs treated cells and tissues were isolated according to the manufacturer’s protocol of DNeasy Blood & Tissue Kit (Qiagen). 200 ng of isolated amplified genomic DNA (20 μl of purified PCR product in 1x NEB 2. Buffer (NEB)) was then denaturated and reannealed to form heteroduplexes by using the next program on Veriti Thermal Cycler (life Technologies): 95° C for 10 min; 95° C to 85° C ramping at –2° C/s; 85° C to 25° C at – 0.25° C/s; and 25° C hold for 1 min. After reannealing process, products were treated with 1 μl of T7 Endonuclease I (NEB) and were analyzed on 10-15 % native PAGE gel. Gels were stained with SYSYBR Gold DNA stain (Life Technologies) for 30 min and were imaged with a DNA bioimaging system (Bio-Rad). Quantification was based on 1/2

relative band intensities. Indel percentage was determined: 100 x (1 – (1 – (b + c)/(a + b + c)) ), where a is the integrated intensity (determined by using ImageJ software) of the undigested PCR product, wherein b and c are integrated intensities of cleaved PCR product. ELISA assays Values of mouse HGF were determined by using Mouse HGF ELISA Kit (Abcam) according to manufacturer’s protocol. Immunoblotting Samples were lysed using SDS (for CD63 under non-reducing conditions; for other proteins SDS with reducing agent was used) and then heat denatured at 95°C for 5 min. Proteins were separated by SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare). Blots were incubated with appropriate antibodies by the use of iBind Western Systems (ThermoFisher Scientific) according to the manufacturers’ protocol. The immunoblots were visualized on G-box (Syngene) after they were developed using Pico or Femto Sensitivity substrate (ThermoFisher Scientific). Electron microscopy EVs were fixed with 4% formaldehyde in 0,1M phosphate buffer (pH7.2). The pellet was washed with PBS/0.15% glycine, embedded in 12% gelatin and cut into 0.5 x 0.5 x 0.5-mm blocks. The blocks were cryoprotected with 2.3 M sucrose, mounted on specimen holders, and frozen in liquid nitrogen. Ultrathin cryosections (50 nm thick) were cut.. Sections were retrieved with a 1:1 mixture of 2.3 M sucrose and 2% methyl cellulose on Au grids. Unspecific labeling was blocked with 1% BSA-c

TM

(Aurion). Sections were incubated with anti-Cas9 antibody and then with protein A conjugated to 18nm gold (UMC Utrecht) and for CD63 with 6-nm gold. Sections were embedded and stained with methyl cellulose/uranyl acetate and examined in a Philips CM100 transmission electron microscope at 80 kV. Animals BALB/c mice were purchased from Harlan (Italy). C57BL/6-Tg(UBC-GFP)30Scha/J (B6-EGFP) mice were purchased from the Jackson Laboratory (USA). Eight- to 12-week-old male and female mice



Page 14 of 25

were used for experiments. All animal experiments were performed according to the directives of the EU 2010/63 and were approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection of the Ministry of Agriculture, Forestry and Foods, Republic of Slovenia (Permit Number U34401-3/2017/8). In vivo luciferase imaging 6

BALB/c mice were injected in the right flank with 2 × 10 HEK293, transfected with the 7sgRNA[B]-minfLuc reporter vector. One hour later, the mice were treated intravenously with 150 μg of EVs containing dCas9-VPR and sgRNA[B]. After 24 h, the mice received 150 mg/kg of body weight of Dluciferin (Xenogen) intraperitoneally and were in vivo imaged with IVIS® Lumina Series III (Perkin Elmer). Data were analyzed with Living Image® 4.5.2 (Perkin Elmer). In a separate experiment, the BALB/c mice were hydrodynamically injected in the tail vein with a volume of saline solution equivalent to 10% of body weight in 4-7 s, containing 60 μg of the 7sgRNA [B]-min-fLuc reporter vector using a 3 ml latex free syringe with a 27 G needle (Beckton Dickinson). Then, 150 μg of EVs carrying dCas9-VPR and sgRNA[B] were intravenously injected into the tail vein. On the following day, the mice received 150 mg/kg of body weight of D-luciferin (Xenogen) intraperitoneally and were in vivo imaged with the IVIS® Lumina Series III (Perkin Elmer). Data were analyzed with Living Image® 4.5.2 (Perkin Elmer). In addition, ex vivo organs were imaged. In vivo tracking of extracellular vesicles GEDEX and ctrl EVs were stained with DiD oil (ThermoFisher Scientific). After the EVs were isolated with ultracentrifugation, 1 μM DiD oil in PBS was added. EVs were stained at room temperature for 10 min protected from light. Then 10 ml PBS was added to the stained EVs, and they were ultracentrifuged for 1 h at 100,000 ×g at 4 °C. One hundred micrograms of stained EVs in a total volume of 100 μl were i/v injected in the tail vein of the BALB/c mice. The mice were then anesthetized with isoflurane, and in vivo fluorescence was determined with the IVIS® Lumina Series III (Perkin Elmer). For the fluorescence measurement, a 640 nm excitation filter and a 670 emission filter were used. Tissue and feed auto-fluorescence was excluded with the spectral un-mixing mode. In addition, ex vivo organs were imaged. Data were analyzed with Living Image® 4.5.2 (Perkin Elmer). Acute liver injury–induced mouse model BALB/c mice were given per os a single dose of 1-naphthyl isothiocyanate (75 mg/kg of body weight; Sigma) dissolved in olive oil (8 ml/kg; Sigma). One hour after ANIT administration, the mice were hydrodynamically injected with 150 μg of EVs carrying sgRNA against mHgf and dCas9-VPR. The mice were hydrodynamically injected in the tail vein with a volume of saline solution equivalent to 10% of body weight in 4-7 s containing EVs using a 3 ml latex-free syringe with a 27 G needle (Beckton Dickinson). Negative control received control EVs after the ANIT oral dose. In a separate experiment, EVs carrying sgRNA against mHgf and dCas9-VPR were injected via a simple i/v injection (the total injected volume was 100 μl). After 48 h, the mice were humanely euthanized, and blood and liver tissue samples were collected for further analysis. Serum was prepared (Sarstedt; 3000 RPM/30 min).


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The mice liver profile was determined in mice sera using the VetScan® Mammalian Liver Profile reagent rotor and analyzed on the biochemistry analyzer VetScan VS2 (Abaxis). Histology of mouse liver Liver tissue samples of mice that underwent hydrodynamic injections were fixated overnight in 10% neutral buffered formalin (Sigma Aldrich) and then embedded in paraffin (Leica Paraplast). The paraffin blocks were cut 7 μm thick with a rotation microtome RM 2245 (Leica). The tissue sections underwent deparaffinization and rehydration using xylene and different dilutions of ethanol (Sigma Aldrich). The tissues samples were then mounted on slides using Leica CV Mount (Leica) and subjected to visualization using confocal microscopy. To determine histopathological changes after the ANIT treatment, tissue sections from the liver tissue samples were H&E stained and then visualized using a Leica DMi8 with a Leica MC170 HD camera. Statistical analyses Data are presented as means ± SD or ± SEM. Representative graphs and images are shown. The Student unpaired two-tailed t test was used for the statistical comparison of the data.

SUPPORTING INFORMATION. Supporting Figures, Methods and tables, including list of primers used in this study and protein concentration compared to particle numbers. AUTHOR INFORMATION Corresponding author *Email: [email protected] Author Contributions R.J. conceived and supervised the study. D.L. and R.J. designed the experiments, with the help of the other authors. D.L., L.K., M.M.K., and I.H.B. performed the experimental work. R.R. performed electron microscopy. All the authors analyzed and discussed the results. D.L. and R.J. wrote the manuscript. All the authors discussed and commented on the manuscript before submission. Notes The authors declare no conflict of interests.

ACKNOWLEDGEMENTS We thank Dr. Jelka Pohar for help with the confocal fluorescence microscopy. We thank Dr. Monika Avbelj for help with isolating the Cas9 protein. We thank Dr. Anže Smole for help with isolating the EVs. We thank Irena Škraba and Luka Brvar for isolating the plasmids. We thank Vesna Mrak and Katja Škulj for help maintaining the mouse colony. We thank Dr. Simon Horvat for help regarding giving oral gavages in mice.



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This work was supported by grants from the Slovenian Research Agency [P4-0176, P3-0108, J3-6791, J1-6740].

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Figure 1. Generation of the GEDEX and its efficiency in functional CRISPR/Cas9 delivery. (A) HEK293 cells were transiently transfected with the Cas9/sgRNA plasmid vector for 24 h. EVs were subjected to immunoblot analysis using antibodies against Cas9, CD63, tsg101(an extracellular vesicle marker) and against calnexin, an apoptotic bodies maker. A representative image from three independent experiments is shown. (B) HEK293, blank and GEDEX producing cells (cells that were transiently transfected with the Cas9 with or without the sgRNA plasmid vector for 24 h) were lysed and immunoblotted against Cas9, tsg101 and calnexin. (C) Extracellular vesicles from non-transfected HEK293 cells (the ctrl GEDEX) and from the Cas9/sgRNA plasmid–transfected cells were subjected to electron microscopy. An arrow shows the stained Cas9 protein within the EV. A representative image is shown. Scale bars, 60 nm. (D) sgRNA fold difference was assessed using qPCR from ctrl GEDEX and from GEDEX carrying Cas9 with/without sgRNA. Cas9 RNP GEDEX was subjected to RNase A treatment before RNA isolation. Data are presented as the mean ± SD. (n=3), statistical analysis with a two-tailed t test (*p

Delivery of an Artificial Transcription Regulator dCas9-VPR by

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