An Embeddable Molecular Code for Lewis X Modification Through Interaction with Fucosyltansferase 9

N-glycans are diversified by a panel of glycosyltransferases in the Golgi, which are supposed to modify various glycoproteins in promiscuous manners, resulting in unpredictable glycosylation profiles in general. In contrast, our previous study showed that fucosyltransferase 9 (FUT9) generates Lewis X glycotopes primarily on lysosome-associated membrane protein 1 (LAMP-1) in neural stem cells. Here, we demonstrate that a contiguous 29-amino acid sequence in the N-terminal domain of LAMP-1 is indispensable for FUT9-dependent Lewis X modification. Interestingly, Lewis X modification was induced on erythropoietin as a model glycoprotein both in vivo and in vitro, just by attaching this sequence to its C-terminus. Based on these results, we conclude that the amino acid sequence from LAMP-1 functions as a “Lewis X code”, which is deciphered by FUT9, and can be embedded into other glycoproteins to evoke a Lewis X modification, opening up new possibilities for protein engineering and cell engineering.

T–Cell Engineering for Chimeric Antigen Receptor T–Cell Therapy in Cancer

Through T-cell engineering, researchers at the California South University (CSU) Cancer Research Institute (CRI) have shown that tumor growth can be stopped in a variety of cancers and prevented from spreading to other tissues. Findings from this study are the result of decades of research by Professor Alireza Haidari, a member of the Cancer Biology Research Program at the California South University (CSU), who discovered a protein called AH that can inhibit the growth and spread of cancer cells in several different ways. They become in the tissues of the body. Keywords: Cancer; Cells; Tissues; Tumors; Prevention; Prognosis; Diagnosis; Imaging; Screening, Treatment; Management

Using chimeric antigen receptor T-cell therapy to fight glioblastoma multiforme: past, present and future developments

Introduction Glioblastoma multiforme (GBM) constitutes one of the deadliest tumors to afflict humans, although it is still considered an orphan disease. Despite testing multiple new and innovative therapies in ongoing clinical trials, the median survival for this type of malignancy is less than two years after initial diagnosis, regardless of therapy. One class of promising new therapies are chimeric antigen receptor T cells or CAR-T which have been shown to be very effective at treating refractory liquid tumors such as B-cell malignancies. However, CAR-T effectivity against solid tumors such as GBM has been limited thus far. Methods A Pubmed, Google Scholar, Directory of Open Access Journals, and Web of Science literature search using the terms chimeric antigen receptor or CAR-T, GBM, solid tumor immunotherapy, immunotherapy, and CAR-T combination was performed for publication dates between January 1987 and November 2021. Results In the current review, we present a comprehensive list of CAR-T cells developed to treat GBM, we describe new possible T-cell engineering strategies against GBM while presenting a short introductory history to the reader regarding the origin(s) of this cutting-edge therapy. We have also compiled a unique list of anti-GBM CAR-Ts with their specific protein sequences and their functions as well as an inventory of clinical trials involving CAR-T and GBM. Conclusions The aim of this review is to introduce the reader to the field of T-cell engineering using CAR-Ts to treat GBM and describe the obstacles that may need to be addressed in order to significantly delay the relentless growth of GBM.

CRISPR-Cas12a Nucleases Function With Structurally Engineered crRNAs – SynThetic trAcrRNA

CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate eleven types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region lead to comparable genome editing efficiencies. In conclusion, we show for the first time that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.

Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

T Cell Engineering as Therapy for Cancer and HIV

Through T-cell engineering, researchers at the California South University (CSU) Cancer Research Institute (CRI) have shown that tumor growth can be stopped in a variety of cancers and prevented from spreading to other tissues. Findings from this study are the result of decades of research by Professor Alireza Heidari, a member of the Cancer Biology Research Program at the California South University (CSU), who discovered a protein called AH that can inhibit the growth and spread of cancer cells in several different ways. They become in the tissues of the body. Keywords: Cancer; Cells; Tissues, Tumors; Prevention, Prognosis; Diagnosis; Imaging; Screening; Treatment; Management

Clinical-Scale Production and Characterization of Ntla-5001 - a Novel Approach to Manufacturing CRISPR/Cas9 Engineered T Cell Therapies

Adoptive T cell therapy has shown exciting efficacy in the treatment of certain hematological malignancies, particularly B cell tumors. However, with other cancers there has been limited success to date, and there remain significant challenges to develop safe and effective advanced cell therapies. Therefore, Intellia Therapeutics is leveraging its proprietary genome editing and cell engineering capabilities to develop a next-generation T cell therapy for the treatment of acute myeloid leukemia (AML). NTLA-5001 is an autologous T cell drug product genetically modified using CRISPR/Cas9 to eliminate endogenous T cell receptor (TCR) expression and transduced with AAV to site-specifically integrate a transgene encoding a Wilms' Tumor 1 (WT1)-targeting TCR into the TRAC locus. The TCR recognizes an HLA-A*02:01 restricted epitope of WT1. To overcome manufacturing difficulties often seen in autologous cell therapies, we developed a robust, electroporation-free, functionally closed-system manufacturing process capable of producing large numbers of minimally differentiated T cells with high editing rates, robust transduction efficiency, low translocation rates, and high viability. The manufacturing process begins with the enrichment of CD8 + and CD4 + T cells from patient apheresis to facilitate an optimum CD8:CD4 ratio at culture initiation. After incubation, T cells are stimulated using an αCD3 αCD28 activation reagent followed by disruption of the TCRβ chain by CRISPR/Cas9 via a lipid nanoparticle (LNP) containing mRNA encoding SpCas9 and sgRNA targeting TRBC. TCRα is subsequently knocked out in the same manner using an LNP containing SpCas9 mRNA and a sgRNA targeting the TRAC locus followed by delivery of the WT1-TCR transgene using AAV-6 for site-specific integration of the WT1-TCR template into the TRAC locus via homology directed repair. T cells are then expanded for several days under constant perfusion using a chemically defined expansion media in a rocking bioreactor until harvest, formulation, and cryopreservation. To date, clinical-scale production of NTLA-5001 at Intellia using healthy donors (n = 6) averaged 9.2 days in length. In that time, the process produced an average of 24.3 × 10 9 total T cells with an average viability of 93%. Although T cells underwent rapid expansion, they retained a minimally differentiated phenotype, with >90% of T cells at harvest expressing CD62L. Using our novel LNP-mediated cell engineering approach, we were able to achieve an average of 98.0% knockout of the endogenous TCR while simultaneously expressing the WT1-TCR in an average of >50% of T cells. This sequential editing approach reduced TRBC/TRAC translocation rates to near background levels. In addition, NTLA-5001 drug product displayed cytotoxic functionality when exposed to cell lines presenting the target WT1 peptide. The NTLA-5001 clinical-scale manufacturing process is a controlled and robust platform for the generation of minimally differentiated T cells with high rates of editing and transgene expression. Disclosures Cossette: Intellia Therapeutics: Current Employment. Aiyer: Intellia Therapeutics: Current Employment. Kimball: Intellia Therapeutics: Current Employment. Luby: Intellia Therapeutics: Current Employment. Zarate: Intellia Therapeutics: Current Employment. Eng: Intellia Therapeutics: Current Employment. Doshi: Intellia Therapeutics: Current Employment. Cole: Intellia Therapeutics: Current Employment. Kolluri: Intellia Therapeutics: Current Employment. Jaligama: Intellia Therapeutics: Current Employment. Gardner: Intellia Therapeutics: Current Employment. Wood: Intellia Therapeutics: Current Employment. Clark: Intellia Therapeutics: Current Employment.