Genetic Engineering Publications - GEG Tech top picks

Immunotherapy, particularly CAR T-Cell cancer therapy, extends the lives of many patients. But sometimes the therapy randomly migrates to places it shouldn't go, sneaking into the lungs or other non-cancerous tissue and causing toxic side effects. However, a team of researchers has discovered the molecule responsible for guiding T cells to tumors, setting the stage for scientists to improve the revolutionary treatment. Their discovery of the crucial migration control gene that expresses ST3GAL1 is the result of "unbiased genomic screening": researchers used a state-of-the-art CRISPR technique to edit thousands of genes expressed in T cells, then tested the migration control capabilities of these genes, one by one over a period of nearly four years, in mouse models. The next step is to find a drug that can manipulate the key T cell protein, ST3GAL1. If the study progresses as planned, such a drug could be added to the CAR T-cell regimen to ensure that the T cells reach their targets

One of the biggest challenges facing cancer researchers is understanding why some patients do not respond to treatment. In some cases, tumours have what is known as multi-drug resistance (MDR), which severely limits treatment options for patients. However, researchers have discovered one of the causes of MDR and a potential strategy to combat it. In their study, the researchers began by using CRISPR technology in mouse stem cells to search for mutations that generate resistance to antitumor agents such as cisplatin, rigosertib or ultraviolet light. Mutations in the FBXW7 gene appeared early on, suggesting that this mutation could confer MDR. In addition, FBXW7-deficient cells showed an excess of mitochondrial proteins, which has already been shown to be associated with drug resistance. Nevertheless, a detailed analysis of these organelles further revealed that the mitochondria of these multi-drug resistant cells appeared to be under severe stress. Finally, the researchers showed that the antibiotic tigecycline is toxic to FBXW7-deficient cells because it activates the 'integrated stress response'. This study thus opens a new avenue of research to combat multidrug resistance.

Three studies reveal that the presence in tumours of two key immune components — B cells and tertiary lymphoid structures — is associated with favourable outcomes when individuals undergo immunotherapy.

In this study, the authors performed an unbiased CRISPR–Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for ‘addiction genes’.  Their results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). In this study, scientists successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. This study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

In the report Kochenderfer et al discuss the efficacy of autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) in patients with relapsed diffuse large B-cell lymphoma.

The immune system can induce metabolic adaptations in tumor cells at an early stage that promote their growth. Researchers identify three proteins that orchestrate this effect: IFNγ, STAT3 and c-Myc. The researchers show that IFNγ activates, in addition to the STAT1-mediated signaling pathway, the STAT3-mediated pathway. This pathway alters the genome expression patterns of the cancer cell by inducing epigenetic changes. It also hyperactivates c-Myc, which is overexpressed in many cancers. The researchers show that genes activated by c-Myc not only shape cancer metabolism, they also compromise T-cell infiltration into tumors and disable their attack on cancer cells. Thus, STAT1- and STAT3-mediated signaling pathways appear to synergize to confer on emerging tumors the ability to avoid immune clearance, resulting in the immunometabolic editing that helps fuel their evolution into full-blown malignancy. The researchers also used CRISPR to screen 2,078 metabolic enzymes in mouse tumors and identified 40 metabolic genes controlled by c-Myc that play an important role in helping cancer cells evade immune surveillance and attack. These enzymes are therefore prime candidates for drug targeting.

Treatments based on immunotherapy give hope to many people with cancer that they will finally be cured. However, some treatments can be very expensive, have side effects, or may only work on a small number of people. That's why researchers at the University of Chicago's Pritzer School of Molecular Engineering have developed a new therapeutic vaccine using a patient's own tumor cells that have been modified to secrete vascular endothelial growth factor and then irradiated so that the cells are dead before being reinjected. This vaccine would therefore train the patient's immune system to detect and eradicate cancer because, according to clinical trials, it stops the growth of melanoma tumors in mouse models. In addition, an immunological memory is set up thanks to the vaccine and leads to long-term effects because the vaccine would destroy the appearance of new tumor cells 10 months after the injection. The injection of the vaccine is done like a traditional vaccine. The advantages of this vaccine are that it would be more effective, less expensive and much safer.

Chimeric antigen receptor (CAR) T cell therapies have impressive activity in the treatment of cancer but are associated with potentially fatal toxicities. In light of the approval of CAR T cell therapy for paediatric patients, a panel of experts from the Hematopoietic Stem Cell Transplantation (HSCT) Subgroup of the Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network, the CAR T Cell Therapy-Associated Toxicity (CARTOX) Program at The University of Texas MD Anderson Cancer Center, and several other institutions have developed consensus guidelines for the use and management of these treatments in paediatric patients, which are presented herein.

In this study, the scientists used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. They generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Their validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and 

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, the scientists devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, they engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus.

The strategy described herein may have broad applications to study cell heterogeneity in human tumors.

Here, the scientists examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Through hydrodynamic tail vein injections, they delivered a PB-CRISPR library into mouse liver. Rapid tumor formation could be observed in less than 2 mo. By sequencing analysis of PB-mediated gRNA insertions, they identified corresponding genes mediating tumorigenesis. Their results demonstrate that PB is a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries for phenotype-driven screens.

In this review, the authors proposed that brain tumor modeling could be well established via CRISPR/Cas9 techniques. CRISPR/Cas9-mediated brain tumor modeling was likely to be more suitable for figuring out the pathogenesis of brain tumors, as CRISPR/Cas9 platform was a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that were closely linked with brain tumors.