Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of certain cancers, including advanced metastatic melanoma, and contributed to significant improvements in patient outcomes. ICIs ...unshackle our immune cells to kill cancer. ICIs that block the interaction of the PD-1 receptor on immune T cells with its ligand PD-L1 on tumour cells have proved particularly successful, with durable responses in approximately 30% of melanoma patients. However, the underlying biology remains poorly understood: PD-L1 expression alone is a poor biomarker for patient response to ICIs and the role of a second PD-1 ligand (PD-L2) in the response to treatment is poorly characterised. Therefore, a fuller understanding of the PD-1 checkpoint pathway is needed to better predict patient response to therapy.
Here we aim to fine-tune endogenous PD-1 and PD-L1/PD-L2 expression in T cells and melanoma respectively, at physiologically relevant levels. We hypothesise that there are optimal expression levels of PD-1/PD-L1 axis proteins which tip the balance towards either immune escape or cancer cell killing. MicroRNA-based fine-tuning of endogenous gene expression is achieved through CRISPR/Cas9-mediated insertion of synthetic microRNA response elements into the 3’UTR of target genes.
Our optimised gene-editing protocols consistently demonstrate highly efficient gene knockout (KO; >80-90%) for a range of targets including PD-1, and PD-L1/PD-L2 in primary human T cells and melanoma cell lines, respectively. We have also achieved up to 98% targeted insertion of a miRNA response element into the target genes PD-1 and PD-L1 in T cells, and a melanoma cell line, respectively. Flow cytometric analysis of PD-L1-edited melanoma cells showed a concurrent decrease in cell surface protein expression indicative of fine-tuned gene expression.
To assess the impacts of melanoma cell PD-L1/PD-L2 modulation on T cell cytotoxic function we co-cultured melanoma-specific T cells with unedited or PD-L1/PD-L2 double-KO melanoma. Preliminary findings in both 2D and 3D spheroid co-culture models indicate that compared to T cells co-cultured with melanoma lacking PD-L1/PD-L2 expression, exposure to PD-1 ligands promoted an altered T cell activation state as demonstrated by decreased co-expression of PD-1 and the activation marker CD137 by flow cytometry. The temporal relationship between this altered T cell activation state and the rate of melanoma cell killing remains to be elucidated.
•We discuss the advantages and disadvantages of AAV as vectors for gene therapy.•We describe the methods for AAV optimization.•The challenges and solutions for delivering CRISPR/Cas9 system using AAV ...as a vectors in gene therapy have been shown in this article.
The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.
Photosynthetic rate at the present atmospheric condition is limited by the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate ...(kcat) and poor affinity for CO2 (Kc) and specificity for CO2 (Sc/o). Rubisco in C4 plants generally shows higher kcat than that in C3 plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C4-type RbcS significantly increased the kcat of Rubisco in a C3 plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C4 plant-like catalytic properties, i.e., higher kcat, higher Kc, and lower Sc/o. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO2 condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the βC-βD hairpin in RbcS. We propose that such differences, particularly in the βC-βD hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties.
Rubisco is a key enzyme of photosynthetic CO2 fixation. Hybrid Rubisco with replacement of rice Rubisco small subunit with sorghum counterpart expressed in rice exhibited C4 plant-like high catalytic activity. Transgenic rice expressing the hybrid Rubisco showed slightly higher photosynthetic capacity. Based on crystal structural analysis, we speculate a possible function of βC-βD hairpin of small subunit in determining the catalytic activity.
Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. ...Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.
CRISPR-Cas9 ribonucleoprotein (RNP) gene editing promises to revolutionize gene manipulation. However, it still lacks an in vivo delivery system and exceeds the loading capacity of delivery vectors, ...limiting its wide range of clinical applications. In this study, we developed cardiac-targeted exosomes loaded with CRISPR-Cas9 RNP to improve gene therapy in the heart for precise genome editing.
Exosomes for gene therapy were isolated from human peripheral blood using ultracentrifugation. We loaded RNP complexes containing Cas9 proteins and miR-34a single guide RNA (sgRNA) into exosomes as therapeutic agents for MI. As a result, gene therapy of the CRISPR/Cas9 RNP complex targeting miRNA-34 in apoptotic cardiomyocytes induced by H2O2 effectively alleviated ischemic damage. To enhance cardiac targeting in vivo, we created a genome editing delivery system by attaching cardiac targeting peptides (CTP) to purified exosomes (CTP-Exo) via click chemistry and loading Cas9 RNPs. Delivery of CTP-Exo@RNP via the tail vein improved bioavailability, with a 1.5-fold increase in delivery to the heart. Importantly, CTP-Exo@RNP exerted a protective effect, attenuating apoptosis to ameliorate myocardial infarction injury. Moreover, CTP-Exo@RNP-treated mice showed a significantly attenuated ejection fraction, fractional shortening, LVEDV, and LVESV compared with those in the MI mice, indicating functional recovery of the MI injury. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting exosomes is a powerful approach for organ-specific gene therapy for cardiovascular disease.
Phospholipids and phytic acid are important phosphorus (P)-containing compounds in rice grains. Phytic acid is considered as a major antinutrient, because the negatively charged phytic acid chelates ...cations, including essential micronutrients, and decreases their bioavailability to human beings and monogastric animals. To gain an insight into the interplay of these two kinds of phosphorus-containing metabolites, we used the CRISPR/Cas9 system to generate mutants of a phospholipase D gene (OsPLDα1) and analyzed the mutational effect on metabolites, including phytic acid in rice grains. Metabolic profiling of two ospldα1 mutants revealed depletion in the phosphatidic acid production and lower accumulation of cytidine diphosphate diacylglycerol and phosphatidylinositol. The mutants also showed significantly reduced phytic acid content as compared to their wild-type parent, and the expression of the key genes involved in the phytic acid biosynthesis was altered in the mutants. These results demonstrate that OsPLDα1 not only plays an important role in phospholipid metabolism but also is involved in phytic acid biosynthesis, most probably through the lipid-dependent pathway, and thus revealed a potential new route to regulate phytic acid biosynthesis in rice.
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide ...RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.
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