Bi-directional gene activation and repression promote ASC differentiation and enhance bone healing in osteoporotic rats.

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

CRISPR activation of long non-coding RNA DANCR promotes bone regeneration.

Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.

CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing.

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

CRISPR technologies for stem cell engineering and regenerative medicine.

CRISPR/Cas9 system exploits the concerted action of Cas9 nuclease and programmable single guide RNA (sgRNA), and has been widely used for genome editing. The Cas9 nuclease activity can be abolished by mutation to yield the catalytically deactivated Cas9 (dCas9). Coupling with the customizable sgRNA for targeting, dCas9 can be fused with transcription repressors to inhibit specific gene expression (CRISPR interference, CRISPRi) or fused with transcription activators to activate the expression of gene of interest (CRISPR activation, CRISPRa). Here we introduce the principles and recent advances of these CRISPR technologies, their delivery vectors and review their applications in stem cell engineering and regenerative medicine. In particular, we focus on in vitro stem cell fate manipulation and in vivo applications such as prevention of retinal and muscular degeneration, neural regeneration, bone regeneration, cartilage tissue engineering, as well as treatment of diseases in blood, skin and liver. Finally, the challenges to translate CRISPR to regenerative medicine and future perspectives are discussed and proposed.

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

Background: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration. Methods: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (BDNF, GDNF and NGF). The hybrid BV was used to transduce rat adipose-derived stem cells (ASC) and functionalize the ASC sheets. We further implanted the ASC sheets into sciatic nerve injury sites in rats. Results: Transduction of rat ASC with the hybrid BV vector enabled robust, simultaneous and prolonged activation of the 3 neurotrophic factors for at least 21 days. The CRISPRa-engineered ASC sheets were able to promote Schwann cell (SC) migration, neuron proliferation and neurite outgrowth in vitro. The CRISPRa-engineered ASC sheets further enhanced in vivo functional recovery, nerve reinnervation, axon regeneration and remyelination. Conclusion: These data collectively implicated the potentials of the hybrid BV-delivered CRISPRa system for multiplexed activation of endogenous neurotrophic factor genes in ASC sheets to promote peripheral nerve regeneration.

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.

CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.

Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells.

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.

Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis.

Rationale: Cisplatin (CDDP) is a broad-spectrum anticancer drug but chemoresistance to CDDP impedes its wide use for cancer therapy. Autophagy is an event occurring in the cytoplasm and cytoplasmic LC3 puncta formation is a hallmark of autophagy. Graphene oxide (GO) is a nanomaterial that provokes autophagy in CT26 colon cancer cells and confers antitumor effects. Here we aimed to evaluate whether combined use of GO with CDDP (GO/CDDP) overcomes chemoresistance in different cancer cells and uncover the underlying mechanism. Methods: We treated different cancer cells with GO/CDDP and evaluated the cytotoxicity, death mechanism, autophagy induction and nuclear entry of CDDP. We further knocked down genes essential for autophagic flux and deciphered which step is critical to nuclear import and cell death. Finally, we performed immunoprecipitation, mass spectrometry and immunofluorescence labeling to evaluate the association of LC3 and CDDP. Results: We uncovered that combination of GO and CDDP (GO/CDDP) promoted the killing of not only CT26 cells, but also ovarian, cervical and prostate cancer cells. In the highly chemosensitized Skov-3 cells, GO/CDDP significantly enhanced concurrent nuclear import of CDDP and autophagy marker LC3 and elevated cell necrosis, which required autophagy initiation and progression but did not necessitate late autophagy events (e.g., autophagosome completion and autolysosome formation). The GO/CDDP-elicited nuclear trafficking and cell death also required importin α/ß, and LC3 also co-migrated with CDDP and histone H1/H4 into the nucleus. In particular, GO/CDDP triggered histone H4 acetylation in the nucleus, which could decondense the chromosome and enable CDDP to more effectively access chromosomal DNA to trigger cell death. Conclusion: These findings shed light on the mechanisms of GO/CDDP-induced chemosensitization and implicate the potential applications of GO/CDDP to treat multiple cancers.

Baculovirus-Mediated miR-214 Knockdown Shifts Osteoporotic ASCs Differentiation and Improves Osteoporotic Bone Defects Repair.

Osteoporotic patients often suffer from bone fracture but its healing is compromised due to impaired osteogenesis potential of bone marrow-derived mesenchymal stem cells (BMSCs). Here we aimed to exploit adipose-derived stem cells from ovariectomized rats (OVX-ASCs) for bone healing. We unraveled that OVX-ASCs highly expressed miR-214 and identified 2 miR-214 targets: CTNNB1 (ß-catenin) and TAB2. We demonstrated that miR-214 targeting of these two genes blocked the Wnt pathway, led to preferable adipogenesis and hindered osteogenesis. As a result, OVX-ASCs implantation into OVX rats failed to heal critical-size metaphyseal bone defects. We further engineered the OVX-ASCs with a novel Cre/loxP-based hybrid baculovirus vector that conferred prolonged expression of miR-214 sponge. Gene delivery for miR-214 sponge expression successfully downregulated miR-214 levels, activated the Wnt pathway, upregulated osteogenic factors ß-catenin/Runx2, downregulated adipogenic factors PPAR-γ and C/EBP-α, shifted the differentiation propensity towards osteogenic lineage, enhanced the osteogenesis of co-cultured OVX-BMSCs, elevated BMP7/osteoprotegerin secretion and hindered exosomal miR-214/osteopontin release. Consequently, implanting the miR-214 sponge-expressing OVX-ASCs tremendously improved bone healing in OVX rats. Co-expression of miR-214 sponge and BMP2 further synergized the OVX-ASCs-mediated bone regeneration in OVX rats. This study implicates the potential of suppressing miR-214 by baculovirus-mediated gene delivery in osteoporotic ASCs for regenerative medicine.

Adipose-derived stem cell sheets functionalized by hybrid baculovirus for prolonged GDNF expression and improved nerve regeneration.

Peripheral nerve regeneration requires coordinated functions of supporting cells (e.g. Schwann cells) and neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF), but nerve regeneration is usually far from complete. Here we constructed a Cre/loxP-based hybrid baculovirus (BV) vector which enabled intracellular formation of episomal DNA minicircle for effective transduction of rat adipose-derived stem cells (ASCs) and prolonged expression of functional GDNF capable of recruiting Schwann cells. The GDNF expression persisted for >20 days with the peak level (≈128 ng/ml) tremendously exceeding the picogram levels of GDNF secreted by neuroprogenitor cells. We further developed a facile method to fabricate and transduce cell sheets composed of undifferentiated ASCs in 2 days, without the need of thermo-responsive polymer commonly used for cell sheet fabrication. Implantation of the hybrid BV-engineered, GDNF-expressing ASCs sheets into sciatic nerve transection site in rats significantly improved the nerve repair, as judged from the enhanced functional recovery, nerve reinnervation, electrophysiological functionality, Schwann cells proliferation/infiltration, axon regeneration, myelination and angiogenesis. The hybrid BV is able to functionalize ASCs sheets by intracellular episomal DNA minicircle formation that circumvents undesired gene integration, and the ASCs sheets fabrication is rapid and simple. These data and features implicate the potentials of ASCs sheets functionalized by the hybrid BV for peripheral nerve regeneration.

Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus.

Calvarial bone repair remains challenging for adults. Although adipose-derived stem cells (ASCs) hold promise to heal bone defects, use of ASCs for critical-size calvarial bone repair is ineffective. Stromal cell-derived factor 1 (SDF-1) is a chemokine capable of triggering stem cell migration. Although recombinant SDF-1 protein is co-delivered with other molecules including BMP-2 to facilitate calvarial bone repair, these approaches did not yield satisfactory healing. This study aimed to exploit a newly developed Cre/loxP-based hybrid baculovirus for efficient gene delivery and prolonged transgene expression in ASCs. We demonstrated that transduction of rat ASCs with the hybrid Cre/loxP-based baculovirus enabled robust and sustained expression of functional BMP-2 and SDF-1. Expression of BMP-2 or SDF-1 alone failed to effectively induce rat ASCs osteogenesis and healing of critical-size calvarial bone defects. Nonetheless, prolonged BMP-2/SDF-1 co-expression in ASCs synergistically activated both Smad and ERK1/2 pathways and hence potentiated the osteogenesis. Consequently, transplantation of the hybrid baculovirus-engineered, BMP-2/SDF-1-expressing ASCs/scaffold constructs potently healed the critical-size (6 mm) calvarial bone defects (filling ≈70% of defect volume), which considerably outperformed the calvarial bone repair using BMP-2/SDF-1 delivered with biomaterial-based scaffolds. These data implicated the potential of Cre/loxP-based hybrid baculovirus vector for ASCs engineering and calvarial bone healing.