Wednesday, February 24, 2021

CRISPR Gene edited ESCs/iPSCs for regenerative and personalized medicine--Ubigene

 


Pluripotent stem cells can give rise to all cells and constitute the mature organism. These specialized cells can be cultured in vitro and are referred to as embryonic stem cells (ESCs). ESCs have undergone a great revolution in developmental biology. These in vitro grown ESCs exhibit the potential to generate all lineages of the embryo in vivo and can give rise to any type of somatic cells such as cardiomyocytes, smooth muscle cells, endothelial cells, neuronal cells and hepatocytes upon in vitro differentiation. The advantages of embryonic stem (ESCs) cells over other cell types are their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Human ESCs gained popularity as a valuable cellular source for the treatment of many degenerative diseases such as ischemic heart failure, Parkinson’s disease, Alzheimer’s disease, diabetes, spinal cord injuries and age-related macular degeneration. In 2010, first time human ESCs were employed to treat spinal cord injuries and there after a dozen clinical trials have been conducted with human ESCs to treat severe ischemic left ventricular dysfunction, age-related macular degeneration, Parkinson’s disease and diabetes, among other degenerative conditions.  

However, the human ESC-based clinical trials suffer immensely from the ethical concerns regarding the use of cells of embryonic origin as well as from failed in vitro fertilized embryos that could result in abnormal development, and from the concerns of immune rejection after transplantation due to the allogenic origin of ESCs. Takahashi and Yamanaka (2016) made a breakthrough discovery of reprograming somatic cells to pluripotent state. In their study, somatic cells such as skin biopsy derived fibroblasts and peripheral blood derived T lymphocytes were reprogrammed through the forced ectopic expression of the transcriptional factors OCT4, SOX2, KLF, c-MYC, NANOG and LIN28. These cells, termed as induced pluripotent stem cells (iPSCs), exhibit similar gene expression, epigenetic profile and the differentiation potential to give rise to any type of somatic cells as that of ESCs.

 Application:

ESCs and iPSCs have wide application in Bio-medical research.

1. Basic Research: Understanding cell fate control, Cell rejuvenation, Studying pluripotency, Tissue and organ development and Physiology.

2. Drug Discovery: Drug discovery for cardiovascular disease, Drug discovery for neurological and neuropsychiatric diseases, Drug discovery for rare diseases etc.

3. Toxicology Studies: Relative use of iPSC-derived cell types in toxicity testing.

4. Disease Modeling: Cardiovascular diseases model, Percent share utilization of iPSC for cardiovascular disease modeling, Proportion of iPSC Sources in cardiac studies, Proportion of vector types used in reprogramming, Proportion of differentiated cardiomyocytes used in disease modeling, iPSC-derived organoids for modeling development and disease, Modeling liver diseases using iPSC-derived Hepatocytes, iPSCs in neurodegenerative disease modeling, and Cancer-derived iPSCs.

5. Cell-Based Therapies: Cell therapy for AMD, Autologous iPSC-RPE for AMD, Allogeneic iPSC-RPE for AMD, iPSC-derived dopaminergic neurons for Parkinson's disease, iPSC-derived NK cells for solid cancers, iPSC-derived cells for GvHD, iPSC-derived cells for spinal cord injury, iPSC-derived cardiomyocytes for ischemic cardiomyopathy.

 CRISPR-U™ gene editing in ESCs and iPSC

The CRISPR system, a potent system for genome editing, used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. The CRISPR system combined with inducible pluripotent stem cells can generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. ESCs and iPSCs were engineered with CRISPR used to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways.  Genome editing in ESCs and iPSCs cellular models can advance research program in the area of functional genomics, signaling pathways, drug discovery, drug response, cancer research and cell therapy etc.

To accelerate ESCs and iPSCs research, Ubigene developed CRISPR-U™ for gene manipulation of ESCs and iPSCs. Thereby, possible to achieve genome editing in ESCs and iPSCs with the utilization of the CRISPR/Cas9 system. The CRISPR-U™ system used for rapid and precise gene editing in ESCs and iPSCs. CRISPR-generated cell models have enabled mechanism and treatment explorations of various diseases like cardiovascular diseases, liver diseases etc. In addition, CRISPR-generated cell models could be coupled with drug discovery, safety pharmacology and other therapeutic studies. Ubigene can customize the gene-editing in ESCs and iPSCs as well as can generate various genes modification in animal models.

 

Figure: CRISPR-U™ customized workflow for engineered ESCs and iPSCs

 Case study 1: (Knockout)

HLA Class I depleted platelet can evade natural killer cell immunity

Platelet transfusion is an essential treatment for patients with thrombocytopenia. Alloimmune platelet transfusion refractoriness (allo-PTR) is observed in approximately 5%–15% of patients who receive platelet transfusion, with the most dominant cause being the production of alloantibodies against human leukocyte antigen class I (HLA-I).  In such HLA-I-mediated allo-PTR, transfused platelets are immediately rejected, except for HLA-I-compatible platelets. However, the need to select compatible donors limits the supply, with the most difficult cases being rare HLA-I types. Human induced pluripotent stem cells (iPSCs) have been extensively studied as an ex vivo source for producing human cells and tissues and iPSC-derived platelets (iPLATs) have the potential to resolve the aforementioned issues in current transfusion systems. In the present study, CRISPR/Cas9 mediated HLA-KO iPLATs were generated by knocking out HLA-I complex molecule β2-microglobulin (B2M). Exon 1 of B2M was selected as the knockout target.  For KO B2M, the researcher constructed sgRNA expression vector (pHL-H1-B2M-sgRNA-mEF1a-RiH), CRISPR/Cas9 expression vector (pHL-UbicP-SphcCas9-iP-A) and transfected MK-iPSCs to generate HLAKO iPLAT.  HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in humanized mice model (Hu-NK-MSTRG) reconstituted with human NK cells. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.

Fig1: Production of HLA-KO iPLATs by Knocking Out β2-Microglobulin in imMKCL


imMKCL was first re-reprogrammed to secondary iPSCs (MK-iPSC), in which B2M was knocking  out. After expansion and matured MK-iPSCs release iPLATs contains Kocking out B2M (A, B). Flow-cytometry analysis of the generated CD41a+ CD42b+ iPLATs and  the cell-surface expression of B2M and of HLA-ABC and HLA-E on imMKCLs, iPLATs, JRC platelets, and K562 cells (C, D and E).

 Case study 2:(Point mutation)

Correction of recessive dystrophic epidermolysis bullosa using iPS cells

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin fragility disorder characterized by blistered skin and mucosa that can be inherited in either a dominant (DDEB) or recessive (RDEB) manner. DEB is caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a crucial protein that forms anchoring fibrils (AFs) that stabilize dermal-epidermal adhesion at the basement membrane zone (BMZ).  Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient’s quality of life. There are currently no therapies approved for the treatment of RDEB. The researcher, used CRISPR system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states and corrected mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). They found that three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2-month post grafting. There findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB. This finding could serve as a foundation to translate this treatment into the clinic.

Biallelic Gene Correction of the Homozygous and Heterozygous Mutations in the COL7A1 Gene by CRISPR/Cas9 Plasmid approach and RNP approach:

The researchers employed two CRISPR-mediated genome editing approach for correction of COL7A1 gene mutation in homozygous and heterozygous state of iPSCs.  They applied biallelic gene correction in homozygous iPSCs with CRISPR/Cas9 plasmid approach. They evaluate the efficacy of gene correction in exon 19 of COL7A1 gene in iPSC-derived cells. They found that 10% of the clones had undergone biallelic correction and 40% of clones had undergone monoallelic correction of the COL7A1 mutation in exon 19. The sequence analysis directed that the target sequence areas were free of off-target mutations. To improve correction efficiency, the researcher used the Cas9 protein and a chemically modified synthetic gRNA as a RNP complex. By using RNP approach, they achieved 58% biallelic and 42% monoallelic correction for the homozygous mutation (c.2470insG) in exon 19 of COL7A1, and 19% biallelic and 48% monoallelic correction for the heterozygous mutation (c.2470insG/ c.3948insT) in exons 19 and 32 of COL7A1, respectively. This strategy creates a “scarless” phenotype without leaving a residual footprint and generate a very high efficiency in single iPSCs without evidence of off-target activity.

 

Fig. 2. Evaluation of CRISPR/Cas9 gene-correction efficiency using plasmid- and protein-based methods in iPS cells. (A) Schematic representation of the CRISPR target site for the homozygous (c.2470insG) mutation in exon 19 of COL7A. (B) Components used for plasmid-based gene-correction strategy. (C) T7E1 assay after gene editing targeting exon 19 of the COL7A1 gene. (D) Sanger sequencing confirmed various genotypes. (E) Schematic representation of CRISPR target site for heterozygous (c.2470insG/c.3948insT) mutations in exon 19 of COL7A. (F) Components used for protein-based gene-correction strategy. (G) Sanger sequencing confirmed various genotypes. (H) Summary of the detected genotypes and efficacy after CRISPR/Cas9 gene editing.


Skin integrity and type VII collagen restoration after grafting onto nude mice

The researcher established protocol for generating keratinocytes (iKCs) and fibroblasts (iFBs) from RDEB patient-derived iPSCs. They observed that functional iKCs need 60 days for maturation invitro and 96% of iPSC-derived KCs (iKCs) expressed keratin 14, and 50% of the 96% had high levels of p63 expression. Besides that, they obtained iFBs from iPSCs after 31 days differentiation. They observed that more than 90% of iPSC-derived iFBs with expression patterns consistent with those of normal human FBs. The 3D skin, derived from gene-corrected iKCs and iFBs, was then grafted onto immunodeficient mice and analyzed 2-month postgrafting. They analyze the expression of C7, epidermal differentiation markers such as keratin 14, keratin 10, loricrin, filaggrin, and vimentin. The iPSC-derived corrected xenografts expressed C7 and fully resemble the WT skin, demonstrating that the gene correction restored the protein function in iPSC-derived iKCs and iFBs. 

Fig. 3. Functional verification of gene-corrected RDEB patient iPSC-derived FBs and HSEs. (A) Western blot (WB) analysis assessing type VII collagen (C7) protein expression and secretion in normal human FBs, wild type iPSC-derived FBs (iPSC WT FB), COL7A1-RDEB homozygous mutant (−/−), and corrected (+/+) iPSCs differentiated to FBs. (B) Thermal stability of C7 was analyzed and quantified by (C) limited trypsin digestion of medium from normal human FBs and iPSC gene-corrected RDEB-derived FBs at increasing temperatures. (D) Generation of 3D HSEs using gene-corrected RDEB patient iPSC-derived KCs and FBs, which were then grafted onto nude mice are histologically comparable to those generated using iPSC WT KCs/FBs, 2-month post grafting. H&E staining revealed normal epidermal and dermal morphology. C7 deposition is demonstrated by immunofluorescence (IF) staining (green signal) 2 month after grafting, using LH7.2 antibody. Additional IF staining was performed for keratin 14, keratin 10, loricrin, filaggrin, and vimentin on corrected, mutant, and WT xenografts. (E) Transmission electron microscopy was performed on positive iPSC WT KCs/FBs skin grafts, negative COL7A1-RDEB homozygous mutant and COL7A1-RDEB gene-corrected KCs/FBs skin grafts, and gene-corrected RDEB skin grafts, 2-month post grafting. The BMZ is indicated by black arrowheads.

 Case study 3: (Knockin)

A novel autologous cell therapy for LGMD2A

Limb girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive inherited disorder and the most common form of LGMD. LGMD2A occurs due to loss of functional Calpain 3 (CAPN3), a skeletal muscle-specific isoform of the calcium-sensitive Calpain cysteine protease family. The researcher used CRISPR-Cas9 mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. A CRISPR mediated gene knockin approach were used to edit iPSCs genome carrying three different CAPN3 mutations, and rescue CAPN3 protein in myotube derivatives in vitro. To corrected the mutation, the researcher used guide RNA sequence that targeting CAPN3-exon 14 (50 -CATCTCCGTGGATCGGCCAG-30) cloned in pX458 vector. HDR donor vector was constructed in the pBluescript plasmid backbone with selection cassette of loxP-flanked GFP-2A-neoR driven by HEF1-eIF4g for positive selection and HSV-tk driven by MC1 promoter for negative selection. The 5 -prime homology arm consisting of intron 13 (950 bp), the knockin insert consisting of exons 15–24 cDNA, and sv40 poly(A) signal sequence were in upstream of GFP-2A-neoR cassette. The 3-prime homology arm consisting of the In14 (950 bp) were downstream of the GFP-2A-neoR cassette. To test the rescue of CAPN3 expression in vivo, transplanted gene-corrected iPSC-derived myogenic progenitors into cardiotoxin-pre-injured tibialis anterior (TA) muscles of C3KO-NSG mice and rescue of the CAPN3 mRNA.

Fig 4: Gene Correction of CAPN3 Mutation in 9015 LGMD2A iPSCs

In this study, the researcher employed a homology-directed repair for gene knockin-based correction of CAPN3 mutations (A). LGMD2A iPSCs gene corrected (C1 and C10) spanning region amplified by genomic PCR. RT-PCR analysis were performed of gene-corrected and uncorrected 9015 iPSC-derived myotubes as well as of unaffected myotubes (control). Rescue of CAPN3 protein expression in gene-corrected (C1 and C10) LGMD2A iPSC-derived myotubes were performed with western blot. In the validation study, the patient’s parents and unrelated control iPSC-derived myotubes were used as reference (D). In addition, autocatalytic activity of CAPN3 were also analyses by western blot, the lysates obtained from myotubes incubated for 0 or 15 min at room temperature (E).


Ubigene's iPSC platform


Ubigene focuses on the optimization of iPSC reprogramming, gene editing and differentiation, and has established a set of mature experimental procedures. With CRIPSR-UTMtechnology, gene editing in iPSC is much accessible. Check out our 100 cell lines that we had successfully modified!

Tuesday, February 23, 2021

[Research Highlight] TXNIP positively regulates the autophagy and apoptosis in the rat müller cell of diabetic retinopathy|Ubigene

 


Diabetic retinopathy (DR) is a microvascular complication of diabetes, threatening the vision of working-age and aged population worldwide, which is preventable [1]. Though various studies have been reported in exploring the mechanism and treatments of DR, there are still many unknowns surrounding the disease which calls for further investigation. DR is a neurovascular disease which damages both the retinal vessels and neural cells [2]. As the primary neuroglia crossing from the outer to the inner layer of neuroretina, müller glia provides nutrition and structural stability, which is vital for the homeostasis of retina [3]. Impairment of müller cell under hyperglycemic condition can certainly induce injury to the neuroretina. Therefore, researches in the relationship between müller glia cell and DR are recommended.


Haocheng Ao from Ophthalmology Center of Sun Yat-sen University published an article "TXNIP regulates the autophagy and apoptosis in the rat muller cell of diabetic retinopathy" in Life Sciences (2020IF:3.647) to investigate the effects of TXNIP on autophagy and apoptosis of Muller cells in diabetic retinopathy rats.


Fig 1


Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to measure the expression level of the targets. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/cas9) method was applied for knockout of TXNIP. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and flow cytometry were utilized to detect the apoptosis. Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cell viability. EdU assay was carried out to measure the cell proliferation ability. Retinal immunohistochemistry, retinal frozen section immunofluorescence as well as the electroretinogram (ERG) recording were implemented to detect the function of the retina.


TXNIP was up-regulated under hyperglycemic condition both in vivo and in vitro. Overexpression of TXNIP activated the autophagy and apoptosis in the rat müller cell. Knockout of TXNIP reduced the autophagy and apoptosis in the rat müller cell under high glucose condition. TXNIP positively regulates autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway. Knockdown of TXNIP improved the visual response to light stimulus of DR.


To confirm the role of TXNIP in rMC-1, researchers designed a lentivirus overexpressing rat gene TXNIP (Fig. 2A, provided by Ubigene). RT-qPCR revealed that TXNIP mRNA level of the overexpression group was significantly higher than that of the control vector group (Fig. 2B). In addition, western blotting showed similar increased expression of TXNIP protein level in overexpression group (Fig. 2C). To evaluate the autophagic flux in rMC-1 under TXNIP overexpressing condition, bafilomycin A1 was applied for blocking autophagic flux. As shown in Fig. 2D, the LC3B-II protein level of TXNIP overexpression group significantly increased with bafilomycin A1, which meant that autophagic flux was enhanced. Besides, the expression of autophagy marker p62 declined while Beclin-1 and Atg12-Atg5 compound increased in the overexpression group. Furthermore, researchers found that the Bcl-2 protein level decreased while Bax and Cleaved Caspase-3 rose in TXNIP overexpression group (Fig. 2E), which suggested that overexpressing TXNIP upregulated apoptosis.


Fig 2


In conclusion, based on the findings of our study, researchers validated the overexpression of TXNIP in vivo and in vitro under hyperglycemic condition, which may serve as a potential biomarker in assessing the severity of diabetic retinopathy. Our study unraveled for the first time that TXNIP positively regulates the autophagy in rat müller cell under high glucose condition by inhibiting the PI3K/AKT/mTOR signaling pathway. Downregulation of TXNIP leads to the reduction of autophagy as well as apoptosis, restoring the cell proliferation and cell viability under high glucose condition, and improves the visual function of diabetic rats. This study sheds light on a potential novel therapeutic approach for DR.


Ubigene provides various types of gene-editing vectors for different species and different virus packaging services, which will easily achieve microbial gene knockout, point mutation and knockin. Vectors and virus offered by Ubigene has been successfully applied in the gene knockout and overexpression on more than 100 cell lines.


Ubigene developed CRISPR-U™ which optimizes eukaryotic cells and animal gene-




editing vectors and processes. The efficiency and accuracy are 10x higher than traditional methods. Contact us immediately to know about your research related services!

Monday, February 22, 2021

CRISPR gene-editing HeLa cells--front fighter of medical breakthroughs|Ubigene

 Henrietta Lacks and Hela cells

In 1952, Henrietta Lack (HeLa) cells became the first human cell line that could grow and divide endlessly in a laboratory, leading scientists to label these cells "immortal". Researchers originally took HeLa cells from an aggressive cervical cancer tumor. These cells continue reproducing with a constant supply of nutrients, they produced a new generation of cells in less than 24 hours. Hela cells, like many tumors, have error-filled genomes, with one or more copies of many chromosomes: a normal cell has 46 chromosomes, where HeLa cells contain 76 to 80 total chromosomes, some of which are heavily mutated. These cells also expressed an over reactive telomerase that rebuilds telomeres after each division, preventing cellular aging and cellular senescence, and allowing perpetual divisions of HeLa cells. HeLa cell line has contributed to many medical breakthroughs, from research on the effects of zero gravity in outer space and the development of the polio vaccine to the study of leukemia, the AIDS virus, and cancer worldwide. Although many other cell lines are in use today, HeLa cells have supported advances in most fields of medical research in the decades since HeLa cells were isolated. HeLa cells have played in some of the major advances in fields such as cancer biology, infectious disease, fundamental microbiology, and many others. Research involving HeLa cells has been described in more than 110,00 scientific publications including three who have been awarded Novel Prize: in 2008 for the discovery that the human papillomavirus (HPV) is a causative agent of cervical cancer,  in 2009 for the discovery of the telomerase enzyme, which protects the telomeres of the chromosome, thus preventing the degradation of the chromosome and in 2014 for advances in live viewing of cellular growth. This staggering number makes it clear just how important these cells have been to research over the past six decades.


The versatility and power of HeLa cells have made them an essential laboratory tool that still continues to offer new clues about the basis of human health and disease. HeLa cell one of the popular cellular model as well as tool for life scientists, who willing to study the mechanism of action of diseases or therapeutically active drug molecules as well as to decipher cell signaling events such as DNA damage repair. Researchers chooses HeLa cell as a tool for multidisciplinary studies due to its immortal cell characteristics, available multi-omics database (genomic, proteomic, and transcriptomic), and easy of to access these databases to design and modify research projects. One of the recent examples of using HeLa cells to study the infectivity of the virus SARS-CoV-2 in humans.  Scientists studying COVID-19 using HeLa cells to determine the receptor the virus uses to enter human cells. The coronavirus study got an unprecedented pace, based on the studies rely on HeLa cells.
 Application:

HeLa cells have evolved into a useful model to study a huge variety of topics. HeLa cells derived from HPV-transformed cervical adenocarcinoma (cancer) cells. They have been used for studies including but by no means limited to those in cancer research. As a tumor model, they have been widely used in studies:


1. Tumor cell migration and invasion

2. Drug development

3. Cell death pathways

 CRISPR-U™ gene editing in HeLa cells

CRISPR-Cas systems have formed a revolutionary genome-editing tool, giving a great impetus to the development of life science and our understanding of life. The CRISPR system used for precise genome editing and can generate gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. HeLa cell is an aggressive cervical cancer cell line. Gene editing in HeLa cell line possible to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. These genomes edited HeLa cell lines will allow investigators to study cancer research, cancer therapeutics, cell death research, functional genomics, signaling pathways, drug discovery, drug response, and cell therapy. Ubigene developed CRISPR-U™ for gene manipulation of HeLa cell line. Thereby, possible to achieve genome editing in HeLa cells with the utilization of the CRISPR/Cas9 system. Ubigene can customize the gene-editing in eukaryotic HeLa cells as well as can generate various genes modification in animal models.

CRISPR knockout hela cell workflow

Figure: CRISPR-U™ customized workflow for engineered HeLa model cells

Ubigene developed CRISPR-U™ which optimizes eukaryotic cells and animal gene-




editing vectors and process. The efficiency and accuracy are 10x higher than 

traditional methods. Contact us immediately to know about your research related services!

 Case study 1: (Knockout)

CRISPR/Cas9 mediated TERT disruption can suppress tumor cell survival

Telomeres are the unique structures in eukaryotic cells that maintain chromosome integrity. Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated in most adult somatic cells. In the somatic cell, TERT is inactivated and their telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere erosion to achieve proliferative immortality. Therefore, inactivating TERT is consider to be a promising means of cancer therapy. The researcher applied CRISPR/Cas9 mediated gene-editing system to target the TERT gene in cancer cells. For this purpose, they selected three different cancer cell lines a cervical cancer cell line (HeLa), a pancreatic cancer cell line (PANC1), and a breast cancer cell line (SUM159). They designed three gRNAs (sg1, sg2, and sg3) targeting the human TERT gene exon 2 (E2), exon4 (E4) and exon 6 (E6), respectively. They further design another two gRNAs (sg4 and sg5) targeting the flanking introns of TERT exon 4 (E4) to generate KO TERT cells. In the haploinsufficiency study, the mutated TERT cells shown lower telomerase activity and shorter telomeres. The cell proliferation measurement, cell culture density, and stronger β-gal staining signals provide evidence that mutant TERT cells suffering severe cellular senescent compare to WTPE Hela cells.  Annexin V and PI staining provide evidence about cellular apoptosis and, the TERT mutant shown higher apoptosis than WTPE Hela cells. The TERT haploinsufficiency in cancer cells leads to retarded growth and enhanced cell death In Vitro.

CRISPR/Cas9 mediated TERT disruption can suppress tumor cell survival

Fig1: Retarded growth and increased cell death in Tert+/− cancer cells. (A) Population doubling time of WT and TERT+/− Hela cells. (B) Light microscopy images of WT and TERT+/− Hela cells at Passage 2 (P2), P5 and P7. (C) β-gal staining of WT and TERT+/− Hela cells. Arrows: example of severely senescent cells. (D) Cell death rates of WT and TERT+/− Hela cells determined by LDH assay. (E) Quantification of flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells in WT and TERT+/− Hela cells. ** p < 0.01.


They further studied on TERT haploinsufficiency has any effects on tumor cell survival in vivo using a tumor xenotransplant nude mice model. Six weeks post-inoculation examined the sizes of the xenografts. As expected, the WTPE Hela cells grew into tumor mass (diameters ~2 cm) in animals; where the TERT mutant Hela cells failed to form any tumor xenotransplant in animals. This result support that Cas9-mediated TERT haploinsufficiency can effectively suppress tumor cell growth in vivo. 


Xenotransplant of WT and TERT+/− Hela cells in nude mice

Fig 2. Xenotransplant of WT and TERT+/− Hela cells in nude mice

 Case study 2:(Point mutation)

CRISPR/Cas9 introduces pathogenic MSH2 point mutations cause differential genomic de-stabilization in human cells

Repeat de-stabilization is variously associated with human disease especially in neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. The MSI+ phenotype is not uniform in human neoplasms. Based on the frequency of microsatellite changes MSI are classified into MSI-H (high) and -L(low). Additionally, MSI also classified their distinct qualitative modes, i.e. Type A and Type B. An earlier study reported that tumors occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumors (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). Lynch syndrome (LS) known as hereditary non-polyposis colorectal cancer (HNPCC), is the most common cause of hereditary colorectal (colon) cancer. Mutation of these genes MLHL, MSH2, MSH6, PMS2, and EPCAM responsible for LS. In the present study, MSH2 mutations reported in Lynch syndrome (LS) were introduced into HeLa cells by using the CRISPR/Cas9 system. The researcher used genome editing vector, pSpCas9(BB)-2A-Puro (pX459), gRNA (designed by CRISPR direct and CRISPR Design Tool) and subcloned into pX459 using the BbsI site. The gRNA-encoding pX459 plasmid and three different single-stranded ssODNs were co-transfected into cells using Lipofectamine 2000. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. However, microsatellites were not markedly destabilized as in MSI-H tumors occurring in LS patients and all the observed alterations were uniformly Type A, which confirms the results in mice. These findings suggest added complexities to the molecular mechanisms underlying repeat destabilization in the human genome.

CRISPR/Cas9 point mutations hela cell case

Fig. CRISPR/Cas9 system introducing MSH2 G674 mutations into HeLa cells. (A) Functional domains, including Walker A motif, in human MSH2 protein, (B) Local DNA and amino acid sequences in Walker A motif in each G674-mutant version, (C) The structure of the guide RNA and ssODNs used for study. (D)Sanger method confirm each of the established HeLa clones.

In this study, MSH2-mutant HeLa clones have been successfully obtained, and the established clones clearly exhibited MMR-defective phenotypes with alkylating agent tolerance and elevated mutation frequencies. Although, in these clones, microsatellites were not markedly destabilized as in tumors occurring in LS patients. The present finding suggested that, in addition to defective MMR, previously unrecognized molecular mechanisms may underlie repeat destabilization in human cells.

 Case study 3:(Knockin)

Reversible cloaking/uncloaking strategy with sgRNAs to control the CRISPR–Cas9 gene editing efficiency in vitro and in living cells

"RNA cloaking" is a mild and reversible chemical approach to controlling RNA hybridization, folding, and enzymatic interactions. In this study, the researcher used an azide-substituted acyl imidazole reagent (NAI-N3) to acylate the 20 -OH groups of RNAs post synthetically, resulting in blocking of the RNAs' folding, hybridization, and function. The activity of such poly-acylated (“cloaked”) RNAs in vitro was efficiently recovered upon treatment with a water-soluble phosphine that triggers Staudinger's reduction of the azide, followed by spontaneous loss of acyl groups (“uncloaking”). The researcher studies these reversible cloaking/uncloaking events with sgRNAs to control the CRISPR-Cas9 gene editing efficiency in vitro and in living cells. For the in-vitro study, they designed an in vitro gene editing model platform with a 103 nucleotide (nt) long synthetic sgRNA targeting a Cy5-labeled double-stranded DNA (dsDNA) target encoding a portion of the green fluorescent protein (GFP). They found that the highest levels of Cas9-mediated DNA cleavage -quantitatively complete restoration of activity to the level of untreated sgRNA-were obtained when the cloaked sgRNA was incubated with tris (hydroxypropyl) phosphine (THPP) or diphenylphosphinobenzene-3-sulfonate (TPPMS) in phosphate-buffered saline (PBS) buffer at 37 C for 1 hour at 1-5 mM concentrations. This strikingly robust performance in vitro directs the possible utility of this method in control of timing and initiation in diagnostic applications of CRISPR-Cas9. For in vivo study, the researcher used GFP-positive HeLa cells that were initially transfected with GFP-targeting sgRNA and Cas9 protein using nucleofection or commercial cationic lipids. They performed an experiment to test whether GFP-targeting sgRNAs that were cloaked based on the same procedure as in the in vitro experiments and delivered by CART were able to block CRISPR–Cas9 genome editing in the GFP-positive HeLa cells. They observed that the GFP-positive cells that were transfected with cloaked sgRNA/ Cas9 mRNA expressed the same level of GFP fluorescence as untreated cells, confirming that acylation of sgRNA blocks essentially all sgRNA activity in live cells. This study highlighted the utility of reversible RNA acylation as a novel method for temporal control of the genome-editing function.

CRISPR/Cas9 knockin hela cell case 1

Fig 1: (a) Mechanism of NAI-N3-enabled inhibition of CRISPR–Cas9 gene editing. sgRNA cloaking inhibits the RNA-guided DNA double strand cleavage by Cas9 nuclease (b) PAGE analysis of Cas9 nuclease assay in vitro using Cy5-labelled dsDNA and untreated, cloaked, and uncloaked (phosphine-treated) sgRNA. (c) Bar chart represents the fraction of cleaved DNA after incubation of the sgRNAs with Cas9.

CRISPR/Cas9 knockin hela cell case 2

Fig 2: Phosphine control of cloaked CRISPR–Cas9 activity in human cells. (a) Flow cytometric analysis of GFP(+) HeLa cells showing loss of activity of cloaked sgRNA, and restoration of editing with phosphine treatment; (b) chart showing gene editing efficiencies (from flow cytometry data) of untreated, cloaked, and phosphine-uncloaked sgRNA (c) epifluorescence microscope images showing GFP knockout upon transfection with untreated sgRNA and Cas9 mRNA, lack of editing activity with cloaked sgRNA leaving GFP fluorescence unaffected, and restoration of editing with phosphine treatment of the cells.

Sunday, February 21, 2021

ARPE-19, Genome Surgery and Vision Science, A combo for tackling blindness--Ubigene

gene knockout ARPE-19 cell


ARPE-19 is a spontaneously arising retinal pigment epithelial (RPE) cell line derived in 1986 by Amy Aotaki-Keen from the normal eyes of a 19-year-old male, who died from head trauma in a motor vehicle accident. These cells form a stable monolayer, which exhibits morphological and functional polarity. ARPE-19 cell line has a visually normal karyotype and expresses RPE-specific markers, the retinal pigment epithelium-specific 65kDa protein (RPE65), and cellular retinaldehyde-binding protein (CRALBP), as has been shown at the mRNA and protein levels, respectively.


ARPE-19 is one of the popular cellular models as well as tools for vision scientists, who are willing to study the occurrence of ophthalmic disorders, mechanism of action of diseases, or therapeutically active drug molecules as well as decipher cell signaling events. Retinal disorders can cause impaired vision, severe vision loss or even blindness. Genetic alterations resulting in a dysfunctional retinal pigment epithelium and/or degenerating photoreceptors cause impaired vision. These juxtaposed cells in the retina of the posterior eye are crucial for the visual cycle or phototransduction. Deficits in these biochemical processes perturb neural processing of images capturing the external environment.


To date, there are more than 260 different genes were identified that cause inherited retinal disorders (IRDs). Each IRD is caused by at least one gene that is not working as it should. IRDs can affect individuals of all ages, can progress at different rate, and are rare. However, many are degenerative, which means that the symptoms of the disease will get worse over time. Common types of IRDs include Leber Congenital Amaurosis (LCA), Retinitis Pigmentosa, Choroideremia, Stargardt’s Disease, and Achromatopsia, etc. Notably, there is a distinct lack of clinically approved pharmacological, cell- or gene-based therapies for inherited retinal disease.


Gene editing technologies are rapidly advancing as a realistic therapeutic option. The ability to strategically edit a patient’s genome can constitute a treatment revolution. However, concerns remain over the safety and efficacy of either transplanting retinal cells following ex vivo gene editing, or with direct gene editing in vivo. Ultimately, further refinements to improve efficacy and safety profiles are paramount for gene editing to emerge as a widely available treatment option.

 Application:

ARPE-19 cell lines application in Vision science.


1. Basic Research: Understanding cell fate control, Studying pluripotency, study retinal cell biology, pathophysiology and pharmacology.

2. Drug Discovery: The most widely used RPE model in ocular drug discovery and drug screening. Drug discovery for diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa, etc.

3. Toxicology Studies: Pharmacokinetics at cellular level, studied uptake of drugs and toxicity testing.

4. Disease Modeling: A useful and convenient model for Diabetic Retinopathy (DR), Macula edema research, age-related macular degeneration (AMD), and retinitis pigmentosa.

  CRISPR-U™ gene editing in APRE-19 cells

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is currently the leading gene-editing technology positioned for therapeutic intervention. Genome editing and the creation of cellular models for ophthalmology can advance research programs in the area of functional genomics, signaling pathways, metabolism, cell death, drug discovery, drug response and cell therapy research, etc. Furthermore, gene editing is emerging as an attractive novel treatment strategy for inherited retinal disease.

Recently, CRISPR/Cas9 was applied to ex-vivo correction of an X-linked retinitis pigmentosa GTPase regulator (RPGR) inherited retinal disorders mutation (IRD) and corrected c.3070G>T mutation. This landmark treatment uses the CRISPR approach to a specific mutation in a gene linked to childhood blindness. This proof-of-concept study demonstrates the capability to repair genes and a treatment milestone can be achieved if these findings are safely and effectively applied to patient eyes. To accelerate ophthalmology and visual sciences research, Ubigene developed CRISPR-U™ for gene manipulation of eukaryotic cells. Thereby, possible to achieve genome editing in APRE-19 cells with the utilization of the CRISPR/Cas9 system. The CRISPR-U™ system is used for rapid and precise gene editing in APRE-19 cells. CRISPR-generated cell models have enabled mechanism explorations of various visually impairs diseases, which are mainly related to retinal pathogenesis, toxicity, polarity studies of proteins, gene delivery, multi-omics, drug screening, and signaling pathways.

Due to the unique features of the human eye and complexities associated with the nature of the disease, animal models fail to mimic all aspects of physiology and pathology of eye diseases. Therefore, cell culture models are useful alternative tools to investigate the physiology and pathology of diseases. Besides, cell culture models are advantageous because they are experimentally controlled systems and so the results are more reproducible than those obtained from animal models. Based on the elucidation of disease mechanisms, CRISPR-generated cell models could be coupled with drug discovery, drug screening and other therapeutic studies. Ubigene can customize the gene-editing in eukaryotic cells as well as can generate various genes modification in animal models.


CRISPR-U™ customized workflow for engineered APRE-19 model cells

Figure: CRISPR-U™ customized workflow for engineered APRE-19 model cells

 Case study 1: Knockout mutation

Prominin-1 act as a regulator in human retinal pigment epithelium

Prominin-1 (Prom1) is a trans-membrane glycoprotein, which is expressed in stem cell lineages, and have a role in cancer stem cell survival. In the visual system, Prom1 is concentrated in the photoreceptor outer segment disc membranes and is thought to play a structural role. The expression of the human dominant Prom1 R373C mutation in mice disrupted photoreceptor disk morphogenesis, suggesting that Prom1 plays an integral role in the structural organization of the outer segment. Also, retinal degeneration has been reported in a spontaneous knock out mouse (Prom1rd19) carrying a point mutation in the Prom1 gene. Human mutations in the Prom1 gene cause Stargardt like and bull’s eye macular dystrophies and retinitis pigmentosa. One critical function performed by the retinal pigments epithelium (RPE) is the phagocytosis and lysosomal degradation of shed photoreceptor outer segment tips. Recent studies have shown that autophagy by the RPE is fundamental to this activity. In this study, the researcher investigates the role of prominin1 in metabolically active cells of retinal pigments epithelium (RPE).

The researcher investigated the functional role and significance of Prom1 in the RPE by using a lentiviral construct overexpressing Prom1 and CRISPR/Cas9 knockout (KO) of Prom1 in the RPE. They demonstrate that Prom1 is a novel and key regulator of autophagosome formation and turnover. Moreover, they reveal a molecular mechanism by which Prom1 regulates autophagy flux in the RPE via p62 and HDAC6 association, and by its ability to inhibit mTORC1 and mTORC2 activities.


Expression and localization of Prom1 in human RPE

Figure 1. Expression and localization of Prom1 in human RPE.


Researchers investigated the expression and localization of Prom1 in both immortalized ARPE-19 cells and primary RPE cultures obtained from donor eyes. They observed similar levels of Prom1 expression were observed in ARPE-19 cells and primary RPE cultures. Immunofluorescence staining of Prom1 and β-catenin expression show that Prom1 is mainly distributed in the cytoplasm and perinuclear regions with some nuclear staining in the ARPE-19 and RPE cultures, which failed to co-localize with peripheral β-catenin. Differentiation of RPE was associated with the increased expression of ZO-1 and β-catenin and a decrease in Prom1 expression, compared to non-differentiated RPE, suggesting a correlation between reduced Prom1 expression and RPE differentiation.


Schematic representation of molecular mechanisms regulating Prom1-dependent autophagy in the RPE

Figure 2: Schematic representation of molecular mechanisms regulating Prom1-dependent autophagy in the RPE

This study demonstrated that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function.

Study summary, Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.

 Case study 2: Point mutation

A study of CRISPR-Cas9- and AON-based approaches to correct the splicing defect of the CLRN1 c.254-649T>G mutation

Usher syndrome (USH) is an autosomal recessive genetic disease characterized by deafness, vision loss due to retinitis pigmentosa (RP), and occasional vestibular dysfunction. In USH type 3 (USH3A), these symptoms occur in a progressive and variable manner. USH3A is commonly associated with mutations in CLRN1, a gene encoding the four-transmembrane-domain protein Clarin-1 of largely unknown function. CLRN1 mutations have an estimated prevalence of 1:100,000 individuals worldwide and recently a novel deep intronic CLRN1 founder mutation (c.254-649T>G) in Saudi Arabia. This splicing mutation generates an aberrant exon, which leads to a frameshift and a premature stop codon. A number of studies identifying deep intronic mutations in known or unknown genes increases, there is an unmet need for developing appropriate treatment strategies for this type of mutations.

In this study, researchers focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect.


CRISPR-Cas9-Mediated CLRN1 Editing

Figure 1. CRISPR-Cas9-Mediated CLRN1 Editing

The c.254-649T>G CLRN1 mutation is located in intron 0b and generates a novel splice donor site (SDS), which results in the inclusion of an aberrant exon in the mature mRNA (Fig.1A). To test the CRISPR-Cas9 efficiency for editing the CLRN1 locus, we designed four single guide RNAs (sgRNAs, g1–g4) targeting regions flanking the mutation (Fig1B). This CRISPR-Cas9 approach showed reasonable gene editing efficiency for the excision of the c.254-649T>G mutation from the native CLRN1 locus in HEK293 cells. The gene editing efficiency was around 25.9% ± 0.41%.


AON-Mediated Correction of CLRN1 mRNA Splicing

Figure 2. AON-Mediated Correction of CLRN1 mRNA Splicing

To test the potential of the antisense oligonucleotides (AONs) for correction of aberrant splicing caused by the c.254-649T>G mutation, the researcher designed five AONs (A1– A5) binding to different regions of the CLRN1 transcript. The use of AONs, particularly A2. (Fig2C, D), achieved a substantial correction of aberrant splicing caused by the c.254-649T>G mutation. AONs therefore another possible strategy, in addition to the CRISPR-Cas9 approach, for treating USH3A patients carrying this mutation.

It is well established that the efficiency of CRISPR-Cas9-mediated gene editing can be cell type-dependent. In this regard, targeting cells that express CLRN1 endogenously would be more therapeutically relevant. Therefore, the researcher tested two different retinal pigment epithelium (RPE)-derived cell lines for CLRN1 expression, i.e., ARPE-19, and human retinal pigment epithelial (hRPE) cells provided by the LMU Eye Hospital (Munich, Germany). The researcher found that the endogenous CLRN1 locus can be efficiently edited via CRISPR-Cas9, when delivered using a rAAV approach.

CRISPR-Cas9-Mediated CLRN1 Gene Editing in Transfected ARPE-19 and rAAV Transduced Human RPE Cells.

Figure 3. CRISPR-Cas9-Mediated CLRN1 Gene Editing in Transfected ARPE-19 and rAAV Transduced Human RPE Cells.


In summary, the therapeutic potential of CRISPR-Cas9 and AON-based strategies to correct the splicing of the CLRN1 c.254- 649T>G mutation, and offers an initial premise for further preclinical development, which could lead to the first clinical trials for USH3A patients.



Ubigene developed CRISPR-U™ which optimizes eukaryotic cells and animal gene-

editing vectors and processes. The efficiency and accuracy are 10x higher than traditional methods. Contact us immediately to know about your research related services!

 

Reference:

Prominin-1 Is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci. Vol 58, 2017.

Antisense Oligonucleotide and CRISPRCas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation. Molecular Therapy: Nucleic Acids Vol. 21, 2020.

Friday, February 19, 2021

How to study protein kinase by CRISPR technology?Ubigene

Protein kinases (PK) and their opponents, phosphatases, have been crucial to many scientists. Approximately 2% of the human genome encodes for PKs known as human “kinome”, consisting of 518 protein kinases and their variants. Protein kinases are key regulators of cell function. They mediate most of the signal transduction in eukaryotic cells and coordinate the activity of multiple cellular processes including metabolism, transcription, cell cycle progression, cell movement, differentiation, and apoptosis. They direct the activity, localization, and overall function of many proteins by modifying protein activity, though adding phosphate groups to their substrate.

Mutations and dysregulation of protein kinases play causal roles in human disease, particularly cancer. Their involvement in multiple aspects of cell biology opens the possibility of developing agonists and antagonists for therapeutic use. A growing interest in developing orally active protein kinase inhibitors has led to the approval of several inhibitors for clinical use. 


Protein kinases are among the largest and most well-studied gene families. Protein phosphorylation plays an essential role in intercellular communication in eukaryotic organisms by mediating signal transduction during development, transcription, immune response, metabolism, apoptosis, and cell differentiation. Aberrant regulation of kinases plays a causal role in many diseases, and the study of these proteins and their functions will contribute to the discovery and development of new therapeutics. kinome allows an accurate study of the kinase‐tumor dependent phenotype and, therefore, has the potential to reveal potential therapeutic targets.



The human protein kinome presents one of the largest protein families that orchestrate functional processes in complex cellular networks, and when perturbed, can cause various cancers. The abundance and diversity of genetic, structural, and biochemical data underlie the complexity of mechanisms by which targeted and personalized drugs can combat mutational profiles in protein kinases. To date, PKs are validated and widely accepted targets for drug discovery, more than 40 drugs have been FDA-approved and are on the market. The majority of compounds address tyrosine kinases, with their main therapeutical applications being cancer and inflammation. Coupled with the evolution of system biology approaches, genomic and proteomic technologies are rapidly identifying and characterizing novel resistance mechanisms to inform the rationale design of personalized kinase drugs. 

 Application:

1. Network: Studying the kinome as a network of kinases or kinomics

2. Disease development: Study on Kinases in disease developments (cancer research kinases, cardiovascular disease research kinases, kinases for nervous system research i.e Alzheimer research kinases, Parkinson research kinases, multiple sclerosis kinases, etc, metabolic disorder research, and respiratory research).

3. Drug discovery: Study of identifying kinase substrates, kinase drug response, drug discovery, mutant kinases & downstream effects, disease mechanism. Drug design by unraveling complex relationships between the robustness of targeted kinase genes and binding specificity of targeted kinase drugs. Develop novel strategies for rationally tailored and robust personalized drug therapies.

4. Disease model: Kinase disease models (Cancer, cardiovascular disease, Alzheimer, research kinases, Parkinson, multiple sclerosis, metabolic, and respiratory, etc).

  Case study:

Serotonin receptor 5A is a potential target for anticancer drug development

Breast tumor-initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. Some small molecules can affect BTIC survival. In this study, the researcher demonstrates that exposure of human breast tumor cells to several structurally unrelated selective antagonists of 5-HT5A reduced BTIC frequency and that this effect was phenocopied by a CRISPR-Cas9-mediated knockout of HTR5A. They used a phosphoproteomic approach to establish that exposure of human breast tumor cells to SB-699551 disrupts signaling via the Gα i/o  a coupled pathway and the PI3K/AKT/mTOR axis, consistent with antagonism of 5-HT5A.  They showed that SB-699551 reduced human breast tumor xenograft growth rate and functioned in concert with docetaxel chemotherapy to shrink the xenografts. Collectively they provide genetic, pharmacological, and phosphoproteomic evidence that 5-HT5A is the likely target of SB-699551 and that selective 5-HT5A antagonists might be developed into a novel class of anti-cancer agents that can be combined with cytotoxic therapies to shrink established breast tumor xenografts.

 

Figure-1: Selective antagonists of 5-HT5A inhibit tumorsphere formation by human breast tumor cells.


The researcher used structurally unrelated 5-HT5A selective antagonists that reduce the frequency of tumorsphere initiating cells in breast cancer cell lines and those derived from human patient-derived tumors (Figure1).

 

Figure 2: SB-699551 affects tumorsphere formation by an irreversible mechanism and targets BTIC.


The researcher observed that SB-699551 inhibits tumorsphere formation by an irreversible mechanism and BTIC frequency per se was affected by exposure to SB-699551, we assessed its capacity to affect tumor initiation using ex vivo assay. They found that xenograft formation significantly delayed in cohorts engrafted with tumor cells exposed to SB-699551 in a dose-dependent fashion as well as xenografts arising from SB-699551-treated tumor cells exhibited a reduced growth rate and mass (Fig 2).

 

Fig 3: SB-699551 signals via canonical Gα i/o coupling and through the PI3K/AKT/mTOR pathway.


To determine the effect of SB-699551 on BTIC survival, cells were treated with the compound and identify the signaling pathways that might be affected in breast tumor cell lines after treatment with the compound. They used the PPA, which measures the phosphorylation status of 43 intracellular proteins using phospho-specific antibodies in a sandwich ELISA format. The phosphoproteins whose abundance was most affected by treatment with SB-699551 was shown in Fig. 3b-c. Interestingly, the decreased phosphorylation of pAKT, pCREB, and pATF1 observed previously was maintained after 24h of SB-699551 treatment (Fig.3f-g).

 

Figure 4: Inducible knockout of HTR5A affects tumorsphere formation and targets BTIC


To investigate the genetic loss of HTR5A phenocopy and their effects in functional assays, we used the CRISPR-Cas9 gene-editing technology to conditionally mutate HTR5A in MCF-7 breast tumor cells. MCF-7 cell lines (2-2, 2-3, and 2-8) were generated by dox-induced Cas9 mediated insertions/deletions (INDELS) in HTR5A (inducible knockout; iKO), as established by next-generation sequencing of genomic DNA (Fig4b). To ensure that BTIC frequency per se was affected by the loss of 5-HT5A activity, they performed ex vivo assays with the MCF-7 NT iKO and HTR5A iKO 2–8 cell line.

 

Figure 5: Treatment with SB-699551 inhibits the growth of human breast tumor xenografts in vivo


SB-699551 reduces the growth rate of human breast tumor xenografts in NOD/SCID mice when administered alone and shrinks the xenografts in combination with docetaxel. Histological examination and TUNEL assays revealed an increase in the frequency of apoptotic tumor cells in the xenografts of mice treated with a combination of both agents. The effect of each compound on tumor growth is consistent with SB-699551 and docetaxel targeting BTIC and their non-tumorigenic progeny respectively (Figure 5).

In summary, SB-699551 reduced 5-HT5A activity by compromising its capacity to signal to downstream effectors known to be dysregulated in breast and other cancers. Thus, 5-HT5A is a suitable molecular target for anticancer drug development.

Ubigene developed CRISPR-U™ which optimizes eukaryotic cells and animal gene-editing vectors and processes. The efficiency and accuracy are 10x higher than traditional methods. Contact us immediately to know about your research related services!

 

Reference:

Gwynne, William D., et al. "Antagonists of the serotonin receptor 5A target human breast tumor initiating cells." BMC cancer 20.1 (2020): 1-17.

[Research highlight]A new target for human endometrial cancer treatment |Ubigene

  Human endometrial cancer (EC) is one of the malignant tumors of the female reproductive tract . With the increase of aging populations, ob...