TABLE 1.
Studies using non‐cell‐based loading strategies.
| Study | EV producing cell | EV validation | EV loading method | Cargo type | Target cells | Target genes | Results | Cell targeting mechanism and in vivo administration |
|---|---|---|---|---|---|---|---|---|
| (Wan et al., 2022) | Hepatic stellate cells (LX‐2) |
DLS TEM Markers: CD63+ TSG101+ GM130‐ |
Electroporation on isolated EVs (20% loading efficiency) |
RNP |
AML‐12 LX‐2 |
PUMA CcnE1 KAT5 |
In vitro gene editing: AML‐12(PUMA): 29.7% indels AML‐12(CcnE1): 25.9‐28.8% indels LX‐2 (KAT5): 20.3 % indels |
– |
| Hepatic cells of APAP‐induced liver injury mice | PUMA |
In vivo gene editing: 26.1 % indels |
Intravenous injection, Tropism |
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| Hepatic cells of CCL4‐induced liver fibrosis mice |
CcnE1 |
In vivo gene editing: 9.7% indels |
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| Hepatic cells of Huh‐7 cell‐induced hepatocellular carcinoma mice | KAT5 |
In vivo gene editing: 21.3% indels |
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| (Kim et al., 2017) | SKOV3 cells (ovarian tumor cell line) |
DLS TEM AFM Markers: CD63+ TSG101+ |
Electroporation on isolated EVs (1.75% loading efficacy) |
Plasmid | SKOV3 cells | PARP‐1 |
In vitro gene editing: 27% indels |
‐ |
| SKOV3 xenograft mice | PARP‐1 |
In vivo gene editing: ‐ Reduced tumor weight and volume ‐ Reduced PARP‐1 protein levels |
Intratumoral or intravenous (tail) injection, Tropism |
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| (Majeau et al., 2022) | Mouse serum |
DLS TEM hs‐FCM Markers: CD9+ |
Lipofectamine CRISPRMAX transfection on isolated EVs |
RNP | Anterior tibia muscle cells in reporter Ai9 mice | Ai9 (2 target sites) |
In vivo gene editing: 8.7% gene deletion |
Intramuscular injection |
| Anterior tibia muscle cells of hDMD/mdx mice with a point mutation in exon 23 of hDMD gene | DMD gene (both intron 22 and 24) |
In vivo gene editing: 13.8% deletion of exon 23+24 |
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| (McAndrews et al., 2021) |
In vitro studies: HEK293T |
NTA FCM Markers: Alix+ CD9+ CD47+ CD63+ CD81+ |
Transfection with Exo‐Fect on isolated EVs | Plasmid | KPC689 cells (murine pancreatic cancer cell line with KRAS mutation) | KrasG12D |
In vitro gene editing: 58% reduced KrasG12D mRNA levels |
– |
| In vivo studies: MSCs | KPC689 cells injected into pancreas of mice |
In vivo gene editing: Statistically insignificantly reduced KrasG12D mRNA expression |
Intravenous and intratumoral injection | |||||
| (Zhuang et al., 2020) | HEK293T |
DLS TEM Markers: CD63+ TSG101+ |
Sonication or repeated freeze‐thaw cycles on isolated EVs (15.3% and 37.6% loading efficiency respectively) | RNP | Human liver organoids (PLOs) from human patient primary liver tumor tissue | WNT10B |
Ex vivo gene editing: TDN‐EVs‐RNP: approx. 28% indels EVs‐RNP: approx. 13% indels |
3D tetrahedral DNA nanostructures (TDNs) targeting cancer cell surface proteins were decorated in the surface of isolated EVs through cholesterol conjugation and a heat‐shock process. Gene editing was close to 5‐fold greater with TDN modified EVs compared to non‐modified EVs. In vivo: Intravenous injection |
| HepG2 xenograft liver tumor in mic |
In vivo gene editing: ‐ Ceased tumor development ‐ Decreased tumor volume Increased ALT enzyme levels with increasing TDN concentration suggesting liver toxicity |
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| Wang et al. (2021) | Human umbilical cord mesenchymal stem cells (HucMSCs) |
NTA TEM Markers: CD9+ CD63+ LaminA‐ |
Electroporation on isolated EVs | Plasmid | RAW264.7 (macrophages) | CCL2 gene (insertion of sTNFR1 gene by HDR) |
In vitro gene insertion: Increased sTNFR1 protein expression |
CAQK peptides were chemically crosslinked to the EV surface targeting activated immune cells at the SCI site. CAQK modified EVs mainly accumulated at the SCI site whereas nonmodified EVs accumulated mainly in the liver. In vivo: Intravenous injection |
| Activated immune cells of the spinal cord injury (SCI) in mice |
In vivo gene insertion: sTNFR1 levels increased by more than 5‐fold |
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| (Xu et al., 2020) |
Anti‐CD19‐CAR‐HEK293T HEK293T |
NTA TEM Markers: AnnexinV+ |
Electroporation on isolated EVs | Plasmid |
Raji cells (Burkitt lymphoma cell line, CD19+) Daudi cells |
MYC oncogene |
In vitro gene editing: Raji cells: 5.71% indels (more than 33.8% cells undergo apoptosis) Daudi cells: 3.85% indels |
AntiCD19‐CAR was incorporated into the EV membrane by plasmid transfection of EV‐donor cells. CD19‐specific CAR‐EVs increased CD19+ Raji cells targeting up to 2‐fold. In vivo: Intracardial or intratumoral injection |
| Raji xenograft NOD/SCID mice |
In vivo gene editing: 1.4‐1.8% indels (cells undergo apoptosis) |
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| (Usman et al., 2018) | Red blood cells (RBC) isolated from group O blood |
NTA TEM Markers: Alix+ TSG101+ Stomatin+ Calnexin‐ |
Electroporation on isolated EVs (18% loading efficiency for Cas9 mRNA) | Plasmid | 293T‐eGFP | EGFP |
In vitro gene editing: 10 % gene knockout |
– |
| Cas9 mRNA and sgRNA | NOMO1‐eGFP cells | EGFP |
In vitro gene editing: NOMO1: 32% of cells had complete loss of eGFP expression |
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| MOLM13 (Leukemia cell line) | mir‐125b‐2 locus |
In vitro gene editing: MOLM13: 98% reduction of miR‐125b expression and 90% reduction of miR‐125a expression |
Density light scatter (DLS), Nanoparticle tracking analysis (NTA), Atomic force microscopy (AFM), Transmission electron microscopy (TEM), High‐sensitivity flow cytometry (hs‐FCM), Flow cytometry (FCM).