Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system.

INTRODUCTION: Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. METHOD: In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. RESULTS: The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. CONCLUSION: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

Cell-Based Therapy Approaches in Treatment of Non-obstructive Azoospermia.

The rate of infertility has globally increased in recent years for a variety of reasons. One of the main causes of infertility in men is azoospermia that is defined by the absence of sperm in the ejaculate and classified into two categories: obstructive azoospermia and non-obstructive azoospermia. In non-obstructive azoospermia, genital ducts are not obstructed, but the testicles do not produce sperm at all, due to various reasons. Non-obstructive azoospermia in most cases has no therapeutic options other than assisted reproductive techniques, which in most cases require sperm donors. Here we discuss cell-based therapy approaches to restore fertility in men with non-obstructive azoospermia including cell-based therapies of non-obstructive azoospermia using regenerative medicine and cell-based therapies of non-obstructive azoospermia by paracrine and anti-inflammatory pathway, technical and ethical challenges for using different cell sources and alternative options will be described, and then the more effectual approaches will be mentioned as future trends.

Potential and challenges of placenta-derived decidua stromal cell therapy in inflammation-associated disorders.

Decidual stromal cells (DSCs) isolated from maternal part of placenta, like mesenchymal stromal cells (MSCs), are able to inhibit alloreactivity in-vitro but in a superior way which makes them an attractive alternative for anti-inflammatory therapies. In alloreactivity, when a strong immune response is developed against alloantigens, DSCs develop an anti-inflammatory environment, both through cell-to-cell contact and soluble factors, to prevent the adverse effects of alloantigens. In alloreactivity-associated inflammation, proinflammatory cytokines can be released and then involved in the up-regulation of inflammatory reactions which is one of the main causes of inflammatory related disorders. According to the preclinical and clinical studies, DSCs could be promising alternatives for the treatment of inflammatory-related diseases for which no definitive and successful treatment has been found yet. Here we first present the DSCs functions in creating the anti-inflammatory environment, their immunomodulatory effects, and their advantages over MSCs. Then, preclinical and clinical studies using DSCs for treatment of inflammatory disease including: graft-versus-host-disease (GVHD) after allogeneic hematopoi-etic stem cell transplantation (Allo-HSCT), COVID-19-associated Acute Respiratory Distress Syndrome (ARDS) and in particular, Infertility-related disorders, are presented. Finally, the challenges of using DSCs in clinical settings will be described.

Nano-SiO2/DBN: an efficacious and reusable catalyst for one-pot synthesis of tetrahydrobenzo[b]pyran derivatives.

BACKGROUND: The nano-sized particles enhance the exposed surface area of the active part of the catalyst, thereby increasing the contact between precursors and catalyst considerably. In this study, nano-SiO2/1,5-diazabicyclo[4.3.0]non-5-en was synthesized, characterized and used as a heterogeneous nanocatalyst for the synthesis of tetrahydrobenzo[b]pyran derivatives. Fourier Transform Infrared Spectroscopy, Field Emission Scanning Electron Microscopy, Brunauer-Emmett-Teller plot, Energy Dispersive X-ray Spectroscopy and Thermo Gravimetric Analysis were used to discern nano-SiO2/1,5-diazabicyclo[4.3.0]non-5-en. RESULTS: Tetrahydrobenzo[b]pyrans were synthesized by using nano-SiO2/1,5-diazabicyclo[4.3.0]non-5-en via one-pot three-component condensation of malononitrile, aldehydes and dimedone in H2O/EtOH at 60 °C. The results indicate that tetrahydrobenzo[b]pyrans were synthesized in good to high yields and short reaction times. CONCLUSIONS: The fundamental privileges of this method are short reaction time, plain procedure, recyclability of catalyst and high yields of products.

The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked-out mice embryo.

Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination-activating gene 1) knocked-out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real-time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked-out blastocyst. Our results showed that the expression of trophectoderm genes (-TEAD-4 and Cdx2), pluripotency genes (Nanog and Oct-4) and imprinting gene (H19) in the Rag1 knocked-out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked-out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.

Utilization of CRISPR/Cas9 gene editing in cellular therapies for lymphoid malignancies.

The ability to change the genetic information of immune cells is a powerful tool for basic and clinical settings. CRISPR/Cas9 gene editing technology by providing an efficient approach has accelerated immune cell therapy of cancers. Lymphoid cancers comprise a wide array of disease including lymphoma, multiple myeloma and lymphocytic leukemia. Here, we review therapeutic applications of the CRISPR/Cas9 technology for immune cell therapy of common lymphoid malignancies. We describe current and future therapeutic application of CRISPR/Cas9 technology with the focus on the production and applications of engineered hematopoietic and immune cells against lymphoid malignancies. Furthermore, we provide an overview of the possible challenges and optimization of CRISPR/Cas9 system for ex- and in vivo applications within recent years.

H19/Igf2 Expression and Methylation of Histone 3 in Mice Chimeric Blastocysts.

BACKGROUND: Currently, the efficient production of chimeric mice and their survival are still challenging. Recent researches have indicated that preimplantation embryo culture media and manipulation lead to abnormal methylation of histone in the H19/Igf2 promotor region and consequently alter their gene expression pattern. This investigation was designed to evaluate the relationship between the methylation state of histone H3 and H19/Igf2 expression in mice chimeric blastocysts. METHODS: Mouse 129/Sv embryonic stem cells (mESCs) expressing the green fluorescent protein (mESCs-GFP) were injected into the perivitelline space of 2.5 days post-coitis (dpc) embryos (C57BL/6) using a micromanipulator. H3K4 and H3K9 methylation, and H19 and Igf2 expression was measured by immunocytochemistry and q-PCR, respectively, in blastocysts. RESULTS: Histone H3 trimethylation in H3K4 and H3K9 in chimeric blastocysts was significantly less and greater, respectively (p< 0.05), than in controls. H19 expression was significantly less (p< 0.05), while Igf2 expression was less, but not significantly so, in chimeric than in control blastocysts. CONCLUSION: Our results showed, that the alteration ofH3K4me3 and H3K9me3 methylation, change H19/Igf2 expression in chimeric blastocysts.

In Vitro Pre-validation of Gene Editing by CRISPR/Cas9 Ribonucleoprotein.

BACKGROUND: The CRISPR/Cas9 genome editing system is a powerful and simple gene editing method. The format of the CRISPR components is one of the important factors in targeting efficiency. Compared to plasmid or mRNA (IVTs) format, using the CRISPR/Cas9 system as Cas9-crRNA-tracrRNA RNP format is more efficient and rapid, especially in minimizing some of the pitfalls of CRISPR-mediated gene editing. In addition to efficient in vivo applications of the CRISPR RNP format in a variety of cell types and organisms, another advantage of this approach is usability for in vitro applications in which the crRNAs in the tracrRNA-crRNA structure guides the Mg2+-dependent RNAdirected DNA endonuclease to introduce double-strand breaks at specific sites in DNA. METHODS: Here, Cas9-crRNA-tracrRNA RNP system was used to test the designed crRNAs for in vitro DNA cleavage by Cas9 protein in RAG1, RAG2 and IL2RG genes. RESULTS: The results of cleavage reveal theCas9-crRNA-tracrRNA RNP system is a rapid and efficient way to pre-validate the efficiency of CRISPR cleavage with crRNAs designed for RAG1, RAG2 and IL2RG genes. CONCLUSION: one step in vitro cleavage of DNA by CRISPR/Cas9 ribonucleoprotein complex can be used to pre-validate the functionality and relative efficiency of CRISPR system for targeting genes.

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9.

BACKGROUND: Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences. OBJECTIVES: The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time. MATERIALS AND METHODS: Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously. RESULTS: CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression. CONCLUSIONS: Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.

CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the Mouse NIH/3T3 Cells.

BACKGROUND: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. METHODS: Four single guide RNAs were designed to target sequences in the coding region of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2. RESULTS: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was detected in about 50% of transfected cells. CONCLUSION: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.

Mosaicism in CRISPR/Cas9-mediated genome editing.

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.

Synthesis and Antimycobacterial Activity of Symmetric Thiocarbohydrazone Derivatives against Mycobacterium bovis BCG.

In this work, we reported the synthesis and evaluation of antimycobacterial and antifungal activity of a series of thiocarbohydrazone derivatives which are thiacetazone congeners. The target compounds were synthesized in superior yields by reacting thiocarbohydrazide with different aromatic aldehydes and methyl ketones. Compounds 8, 19 and 25 were found to be the most potent derivatives, exhibiting acceptable activity against Mycobacterium bovis BCG compared to thiacetazone and ethambutol as reference substances. Compounds 8, 15 and 25 exhibited the highest activity against Candida albicans. The most active compounds had a completely different aromatic ring system with various electronic, steric and lipophilic natures. This is understandable in light of the fact that carbohydrazone derivatives must undergo a metabolic activation step before exerting their anti-TB activity and different SAR rules govern each one of these two processes.

Membrane-active metabolites produced by soil actinomycetes using chromatic phospholipid/polydiacetylene vesicles.

Increased resistance of pathogens toward existing antibiotics has compelled the research efforts to introduce new antimicrobial substances. Drugs with new and less resistant-prone targets to antimicrobial activity have a high priority for drug development activities. Cell membrane seems to be a potential target for new antibiotic agent development to overcome resistance. In this study, A total number of 67 actinomycetes were isolated from the soil samples collected from desert, farming and mineral parts of Iran. We used a chromatic sensor as a membrane model that was set up for the target of antimicrobial metabolites of actinomycetes isolated from the soil. The sensors particles were composed of phospholipid and polymerized polydiacetylene (PDA) lipids. These polymers exhibited color change following interaction with membrane-active metabolites. The color change was due to structural disorder in the lipids following their interaction with membrane-active metabolites. The resultant color change was recorded by fluorescent microscope and easily recognizable by naked eye as well. Sixteen strains were isolated which produced antimicrobial metabolites and were effective against test microorganisms (Escherichia coli, Candida albicans and Saccharomyces cerevisiae ). A total number of 3 out of 16 strains produced membrane-active metabolites. These 3 strains were identified using 16s rRNA as Streptomyces sp and submitted to GenBank (accession no. JN180853; JN180854; JN180855).