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A multifunctional non-viral vector for the delivery of MTH1-targeted CRISPR/Cas9 system for non-small cell lung cancer therapy.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.
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Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation.
While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.
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Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.
The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.
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Delivery of gene editing therapeutics.
For the past decades, gene editing demonstrated the potential to attenuate each of the root causes of genetic, infectious, immune, cancerous, and degenerative disorders. More recently, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) editing proved effective for editing genomic, cancerous, or microbial DNA to limit disease onset or spread. However, the strategies to deliver CRISPR-Cas9 cargos and elicit protective immune responses requires safe delivery to disease targeted cells and tissues. While viral vector-based systems and viral particles demonstrate high efficiency and stable transgene expression, each are limited in their packaging capacities and secondary untoward immune responses. In contrast, the nonviral vector lipid nanoparticles were successfully used for as vaccine and therapeutic deliverables. Herein, we highlight each available gene delivery systems for treating and preventing a broad range of infectious, inflammatory, genetic, and degenerative diseases. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 gene editing for disease treatment and prevention is an emerging field that can change the outcome of many chronic debilitating disorders.
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Hematopoietic stem cell therapy with gene modification to treat sickle cell disease.
Hematopoietic stem cells (HSCs) reconstitute blood cells throughout life. DNA-level correction of HSCs allows for a one-time cure of genetic diseases, including sickle cell disease (SCD). Sickle cell disease is one of the most common single-gene disorders; therefore, SCD is a prime candidate for gene therapy. Several drug therapies are available for SCD, including hydroxyurea, which is the first-line choice despite requiring lifelong administration. Allogeneic HSC transplantation is a one-time, curative treatment for SCD with limited availability of histocompatible donors. Therefore, autologous HSC gene therapy was developed using patients' own HSCs with lentiviral gene addition/silencing and clustered regularly interspaced short palindromic repeats gene editing, making gene therapy applicable to most patients. However, the established method of HSC gene therapy requires costly and complex ex vivo HSC culture. Therefore, in vivo HSC gene therapy is being developed to treat SCD, envisioning a single-injection HSC-targeted gene delivery system. This review discusses various therapeutic methods to treat SCD, the development of HSC gene therapy, and clinical gene therapy trials in SCD, ranging from FDA-approved to novel in vivo gene therapy.
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Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia.
BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.
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CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.
Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.
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Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells.
The β2 adrenergic receptor (β2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following β2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β2AR activation. Consistent with this mechanism, β2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the β2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that β2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.
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Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.
BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.
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Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis.
The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.
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