Injectable Supramolecular Hydrogels for In Situ Programming of Car-T Cells toward Solid Tumor Immunotherapy.

Chimeric antigen receptor (CAR)-T cell immunotherapy is approved in the treatment of hematological malignancies, but remains far from satisfactory in solid tumor treatment due to inadequate intra-tumor CAR-T cell infiltration. Herein, an injectable supramolecular hydrogel system, based on self-assembly between cationic polymer mPEG-PCL-PEI (PPP) conjugated with T cell targeting anti-CD3e f(ab')2 fragment and α-cyclodextrin (α-CD), is designed to load plasmid CAR (pCAR) with a T cell specific CD2 promoter, which successfully achieves in situ fabrication and effective accumulation of CAR-T cells at the tumor site in humanized mice models. More importantly, due to this tumor microenvironment reprogramming, secretion of cellular inflammatory cytokines (interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) or tumor killer protein granzyme B is significantly promoted, which reverses the immunosuppressive microenvironment and significantly enhances the intra-tumor CAR-T cells and cytotoxic T cells infiltration. To the best of the current knowledge, this is a pioneer report of using injectable supramolecular hydrogel for in situ reprogramming CAR-T cells, which might be beneficial for solid tumor CAR-T immunotherapy.

Non-invasive activation of intratumoural gene editing for improved adoptive T-cell therapy in solid tumours.

Adoptive T-cell therapy against solid tumours is limited by the apoptosis resistance mechanisms of tumour cells and by the extracellular, immunosuppressive tumour microenvironment. Here we report a temperature-sensitive genome-editing nanodevice that can deliver a Cas9 editor with an external trigger which can be used to edit the genome of tumour cells to reduce resistance to apoptosis and modulate the tumour microenvironment via a mild heating trigger. After local or systemic delivery of Cas9, mild heating is induced by non-invasive near-infrared (NIR) light or focused ultrasound (FUS) to activate Cas9, which initiates simultaneous genome editing of HSP70 (HSPA1A) and BAG3 in tumour cells. This disrupts the apoptotic resistance machinery of the tumour cells against adoptive T cells. At the same time, an NIR- or FUS-induced mild thermal effect reshapes the extracellular tumour microenvironment by disrupting the physical barriers and immune suppression. This facilitates the infiltration of adoptive T cells and enhances their therapeutic activity. Mild thermal Cas9 delivery is demonstrated in different murine tumour models which mimic a range of clinical indications, including a tumour model based on humanized patient-derived xenografts. As a result, the non-invasive thermal delivery of Cas9 significantly enhances the therapeutic efficacies of tumour-infiltrating lymphocytes and chimeric antigen receptor T and shows potential for clinical application.

CRISPR-Based KCC2 Upregulation Attenuates Drug-Resistant Seizure in Mouse Models of Epilepsy.

OBJECTIVE: The precise intervention of K-Cl cotransporter isoform 2 (KCC2) as a promising target for drug-resistant epilepsy remains elusive. METHODS: Here, we used a CRISPRa system delivered by adeno-associated viruses to specifically upregulate KCC2 in the subiculum to confirm its therapeutic potential in various in vivo epilepsy models. Calcium fiber photometry was used to reveal the role of KCC2 in the restoration of impaired GABAergic inhibition. RESULTS: CRISPRa system effectively upregulated KCC2 expression both in in vitro cell culture and in vivo brain region. Delivery of CRISPRa with adeno-associated viruses resulted in upregulating the subicular KCC2 level, contributing to alleviating the severity of hippocampal seizure and facilitating the anti-seizure effect of diazepam in a hippocampal kindling model. In a kainic acid-induced epilepticus status model, KCC2 upregulation greatly increased the termination percentage of diazepam-resistant epilepticus status with the broadened therapeutic window. More importantly, KCC2 upregulation attenuated valproate-resistant spontaneous seizure in a kainic acid-induced chronic epilepsy model. Finally, calcium fiber photometry showed CRISPRa-mediated KCC2 upregulation partially restored the impaired GABAA -mediated inhibition in epilepsy. INTERPRETATION: These results showed the translational potential of adeno-associated viruses-mediated delivery of CRISPRa for treating neurological disorders by modulating abnormal gene expression that is directly associated with neuronal excitability, validating KCC2 as a promising therapeutic target for treating drug-resistant epilepsy. ANN NEUROL 2023;94:91-105.

Genome-editing prodrug: Targeted delivery and conditional stabilization of CRISPR-Cas9 for precision therapy of inflammatory disease.

Regulation of CRISPR-Cas9 functions in vivo is conducive to developing precise therapeutic genome editing. Here, we report a CRISPR-Cas9 prodrug nanosystem (termed NanoProCas9), which combines the targeted delivery and the conditional activation of CRISPR-Cas9 for the precision therapy of inflammatory bowel disease. NanoProCas9 is composed of (i) cationic poly(ß-amino ester) (PBAE) capable of complexing plasmid DNA encoding destabilized Cas9 (dsCas9) nuclease, (ii) a layer of biomimetic cell membrane coated on PBAE/plasmid nanocomplexes for the targeted delivery of PBAE/dsCas9 complexes, and (iii) the stimuli-responsive precursory molecules anchored on the exofacial membrane. The systemic administration of NanoProCas9 enables the targeted delivery of dsCas9 plasmid into inflammatory lesions, where the precursory small molecule can be activated by ROS signals to stabilize expressed dsCas9, thereby activating Cas9 function for inflammatory genome editing. The proposed "genome-editing prodrug" presents a proof-of-concept example to precisely regulate CRISPR-Cas9 functions by virtue of particular pathological stimuli in vivo.

Delivery of a system xc- inhibitor by a redox-responsive levodopa prodrug nanoassembly for combination ferrotherapy.

A comprehensive understanding of ferroptosis signaling pathways significantly contributes to the advances in cancer ferrotherapy. Herein, we constructed a self-assembled prodrug nanosystem targeting system xc-, a key regulator for ferroptosis, to amplify the therapeutic efficacy of cancer ferrotherapy. The prodrug nanosystem is assembled between sulfasalazine (SSZ, a ferroptosis resistance inhibitor) and disulfide-bridged levodopa (DSSD) that can chelate Fe2+ ions to form SSZ-Fe2+@DSSD, and the resulting nanoassembly can not only inhibit ferroptosis resistance, but also generate ROS in the tumor microenvironment. Whereas the prodrug nanosystem is stable in the physiological environment, it becomes unstable in the tumoral and intracellular reductive microenvironment, where the disulfide linkers are disrupted by high levels of glutathione (GSH), triggering the release of active Fe2+ and SSZ. Under the Fenton reaction, the released Fe2+ thus can induce ferroptosis, which is amplified by SSZ-mediated inhibition of ferroptosis resistance to synergistically improve the therapeutic efficacy of ferroptosis. Our study thus provides an innovative prodrug strategy to advance anticancer ferroptosis.

Rational Design of Poly(disulfide)s as a Universal Platform for Delivery of CRISPR-Cas9 Machineries toward Therapeutic Genome Editing.

We synthesized a series of poly(disulfide)s by ring-opening polymerization and demonstrated that the copolymerization of monomer 1 containing diethylenetriamine moieties and monomer 2 containing guanidyl ligands could generate an efficient delivery platform for different forms of CRISPR-Cas9-based genome editors, including plasmid, mRNA, and protein. The excellent delivery performance of designed poly(disulfide)s stems from their delicate molecular structures to interact with genome-editing biomacromolecules, unique delivery pathways to mediate the cellular uptake of CRISPR-Cas9 cargoes, and strong ability to escape the endosome. The degradation of poly(disulfide)s by intracellular glutathione not only promotes the timely release of CRISPR-Cas9 machineries into the cytosol but also minimizes the cytotoxicity that nondegradable polymeric carriers often encounter. These merits collectively account for the excellent ability of poly(disulfide)s to mediate different forms of CRISPR-Cas9 for their efficient genome-editing activities in vitro and in vivo.

Reprogramming the Tumor Microenvironment through Second-Near-Infrared-Window Photothermal Genome Editing of PD-L1 Mediated by Supramolecular Gold Nanorods for Enhanced Cancer Immunotherapy.

A photothermal genome-editing strategy is described to improve immune checkpoint blockade (ICB) therapy by CRISPR/Cas9-mediated disruption of PD-L1 and mild-hyperthermia-induced activation of immunogenic cell death (ICD). This strategy relies on a supramolecular cationic gold nanorod that not only serves as a carrier to deliver CRISPR/Cas9 targeting PD-L1, but also harvests the second near-infrared-window (NIR-II) light and converts into mild hyperthermia to induce both ICD and gene expression of Cas9. The genomic disruption of PD-L1 significantly augments ICB therapy by improving the conversion of dendritic cells to T cells, followed by promoting the infiltration of cytotoxic T lymphocytes into tumors, thereby reprogramming immunosuppressive tumor microenvironment into immunoactive one. Such a therapeutic modality greatly inhibits the activity of primary and metastatic tumors and exhibits long-term immune memory effects against both rechallenged and recurrent tumors. The current therapeutic strategy for synergistic PD-L1 disruption and ICD activation represents an appealing way for cancer immunotherapy.

Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing.

We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.

Delivery of CRISPR/Cas9 for therapeutic genome editing.

The clustered, regularly-interspaced, short palindromic repeat (CRISPR)-associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome-editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome-editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off-target effects. Recently, non-viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non-viral vectors, including polymers, liposomes, cell-penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non-viral vectors are also discussed.

Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Targeted integration of genes in Xenopus tropicalis.

With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.

Role of BmDredd during Apoptosis of Silk Gland in Silkworm, Bombyx mori.

Silk glands (SGs) undergo massive apoptosis driven degeneration during the larval-pupal transformation. To better understand this event on molecular level, we investigated the expression of apoptosis-related genes across the developmental transition period that spans day 4 in the fifth instar Bombyx mori larvae to day 2 pupae. Increases in the expression of BmDredd (an initiator caspase homolog) closely followed the highest BmEcR expression and resembled the expression trend of BmIcE. Simultaneously, we found that BmDredd expression was significantly higher in SG compared to other tissues at 18 h post-spinning, but reduced following injection of the apoptosis inhibitor (Z-DEVD-fmk). Furthermore, BmDredd expression correlated with changes of caspase3-like activities in SG and RNAi-mediated knockdown of BmDredd delayed SG apoptosis. Moreover, caspase3-like activity was increased in SG by overexpression of BmDredd. Taken together, the results suggest that BmDredd plays a critical role in SG apoptosis.

BMDREDD REGULATES THE APOPTOSIS COORDINATING WITH BMDAXX, BMCIDE-B, BMFADD, AND BMCREB IN BMN CELLS.

The apoptosis mechanisms in mammals were investigated relatively clearly. However, little is known about how apoptosis is achieved at a molecular level in silkworm cells. We cloned a caspase homologous gene named BmDredd (where Bm is Bombyx mori and Dredd is death-related ced-3/Nedd2-like caspase) in BmN cells from the ovary of Bm and analyzed its biological information. We constructed the N-terminal, C-terminal, and overexpression vector of BmDredd, respectively. Our results showed that the transcriptional expression level of BmDredd was increased in the apoptotic BmN cells. Furthermore, overexpression of BmDredd increased the caspase-3/7 activity. Simultaneously, RNAi of BmDredd could save BmN cells from apoptosis. The immunofluorescence study showed that BmDredd located at the cytoplasm in normal cell otherwise is found at the nucleus when cells undergo apoptosis. Moreover, we quantified the transcriptional expressions of apoptosis-related genes including BmDredd, BmDaxx (where Daxx is death-domain associated protein), BmCide-b (where Cide-b is cell death inducing DFF45-like effector), BmFadd (Fadd is fas-associated via death domain), and BmCreb (where Creb is cAMP-response element binding protein) in BmN cells with dsRNA interferences to detect the molecular mechanism of apoptosis. In conclusion, BmDredd may function for promoting apoptosis and there are various regulatory interactions among these apoptosis-related genes.

BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx mori.

PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemocytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa protein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva implied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.

TRANSCRIPTION FACTOR Bmsage PLAYS A CRUCIAL ROLE IN SILK GLAND GENERATION IN SILKWORM, Bombyx mori.

Salivary gland secretion is altered in Drosophila embryos with loss of function of the sage gene. Saliva has a reduced volume and an increased electron density according to transmission electron microscopy, resulting in regions of tube dilation and constriction with intermittent tube closure. However, the precise functions of Bmsage in silkworm (Bombyx mori) are unknown, although its sequence had been deposited in SilkDB. From this, Bmsage is inferred to be a transcription factor that regulates the synthesis of silk fibroin and interacts with another silk gland-specific transcription factor, namely, silk gland factor-1. In this study, we introduced a germline mutation of Bmsage using the Cas9/sgRNA system, a genome-editing technology, resulting in deletion of Bmsage from the genome of B. mori. Of the 15 tested samples, seven displayed alterations at the target site. The mutagenesis efficiency was about 46.7% and there were no obvious off-target effects. In the screened homozygous mutants, silk glands developed poorly and the middle and posterior silk glands (MSG and PSG) were absent, which was significantly different from the wild type. The offspring of G0 mosaic silkworms had indel mutations causing 2- or 9-bp deletions at the target site, but exhibited the same abnormal silk gland structure. Mutant larvae containing different open-reading frames of Bmsage had the same silk gland phenotype. This illustrated that the mutant phenotype was due to Bmsage knockout. We conclude that Bmsage participates in embryonic development of the silk gland.

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

Hedgehog signaling pathway regulated the target genes for adipogenesis in silkworm Bombyx mori.

Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the pathway in adipose development remains poorly understood. In this report, we found that Hh pathway components are expressed in the fat body of silkworm larvae. Functional analysis of these components in a BmN cell line model revealed that activation of the Hh gene stimulated transcription of Hh pathway components, but inhibited the expression of the adipose marker gene AP2. Conversely, specific RNA interference-mediated knockdown of Hh resulted in increased AP2 expression. This further showed the regulation of Hh signal on the adipose marker gene. In silkworm larval models, enhanced adipocyte differentiation and an increase in adipocyte cell size were observed in silkworms that had been treated with a specific Hh signaling pathway antagonist, cyclopamine. The fat-body-specific Hh blockade tests were consistent with Hh signaling inhibiting silkworm adipogenesis. Our results indicate that the role of Hh signaling in inhibiting fat formation is conserved in vertebrates and invertebrates.

Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori.

We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7-35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.