Genetic Engineering Publications - GEG Tech top picks

CAR T cells have become an established treatment option for children with a rare form of relapsed or incurable leukemia. One of the key factors determining whether treatment will lead to lasting remission of leukemia is how long the CAR T cells live in the body. One team was able to study the cells of 10 children enrolled in a pioneering clinical trial (CARPALL) for up to five years after their initial CAR T cell treatment. This has enabled them to better understand why some of these CAR T cells remain in a patient's bloodstream, and why others disappear early, potentially allowing the cancer to recur. Using techniques that analyze individual cells at the genetic level to understand what they do, the scientists were able to identify a unique "signature" in long-lived CAR T cells. The signature suggested that long-lived CAR T cells in the blood transformed into a different state that allowed them to continue monitoring the patient's body for cancer cells. As part of the study, the researchers identified key genes in CAR T cells that appeared to enable them to persist in the body for a long time. These genes will provide a starting point for future studies to identify markers of persistence in CAR T-cell products as they are manufactured, and ultimately to improve their efficacy.

A new approach to CRISPR-Cas9 technology allows scientists to introduce particularly long DNA sequences at precise locations in cell genomes at remarkably high efficiencies without the viral delivery systems traditionally used to transport DNA into cells. The researchers found that high levels of double-stranded DNA template can be toxic to cells, resulting in low efficiency. The team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations. The researchers therefore describe in the paper published in the journal Nature Biotechnology, a method for attaching the modified Cas9 enzyme to a single-stranded template DNA, by adding just a small overhang of double-stranded DNA at the ends. The single-stranded template DNA could more than double the efficiency of gene editing compared to the old double-stranded approach. In addition, the double-stranded ends of the molecules allow the researchers to use Cas9 to enhance the delivery of non-viral vectors into cells. In the study, the researchers used the new DNA template to generate more than one billion CAR-T cells targeting multiple myeloma and showed that their approach could, for the first time, replace two genes associated with rare genetic immune diseases, IL2RA and CTLA4, in their entirety

For 17 years, Thomas Blankenstein has been working on his Berlin-based start-up T-knife and is finally going to be able to test his T-cell receptor (TCR) cancer immunotherapy. To make this immunotherapy, they replaced the TCR genes of a mouse with those of a human and inserted genes to encompass both the human TCR repertoire and human major histocompatibility complex (MHC) molecules. They eliminated genes from related mice, generating mouse lines carrying human genes, and then subjected them to a succession of crosses to bring all the genes together in a single mouse, the HuTCR mouse. Designing transgenic mice with such efficient humanized TCRs has been very difficult and laborious. However, TCR-T cells are capable of more extensive signaling and killing than CAR-T cells. The engineered TCRs integrate seamlessly into the signal transduction pathways of T cells: there are ten subunits in a TCR versus one subunit in a CAR, they have ten immunoreceptor tyrosine activation motifs versus three in a CAR, and they are associated with more co-stimulatory receptors: CD3, CD4, CD2. In addition, unlike CARs that only bind to antigens on the cell surface, TCRs can target intracellular as well as extracellular tumor antigens.

A new approach to CAR T cells therapy developed by a UCLA research team allows for an effective and long-lasting response to HIV, unlike current applications of such therapies that cannot provide lasting immunity, i.e., they cannot destroy malignant or infected cells that appear months or years after treatment. Previously, researchers had created a therapy based on CAR T cells containing part of the CD4 molecule because HIV binds to CD4 molecules in order to infect cells in the body. HIV binds to the CD4 molecule of the CAR T cell, which activates it and the cell kills HIV. However, two domains of CD4 molecules still allowed HIV to infect cells. So, the researchers removed these domains and added another that makes the cells resistant to infection and allows a more effective and durable cellular response against HIV than the former therapy. This new CAR T cells therapy with a truncated form of the CD4 molecule works like a vaccine. It stimulates the patient's immune system before HIV itself induces a response. In addition, this therapy induces the production of a large number of memory T cells and could influence the field of immunotherapy focused on engineering T cells with CAR molecules.

A recent study published in the journal Nature Medicine, led by researchers James Riley, PhD, a professor of Microbiology at the Perelman School of Medicine at the University of Pennsylvania, and Todd Allen, PhD, a professor of Medicine at Harvard Medical School and Group Leader at the Ragon Institute of MGH, MIT and Harvard, describes a new Dual CAR T cell immunotherapy that can help fight HIV infection.

Researchers have developed a universal receptor system that allows T cells to recognize any cell surface target, enabling highly customizable CAR T cell and other immunotherapies for treating cancer and other diseases. The new approach involves engineering T cells with receptors bearing a universal "SNAPtag" that fuses with antibodies targeting different proteins. By tweaking the type or dose of these antibodies, treatments could be tailored for optimal immune responses. The researchers showed that their SNAP approach works in two important receptors: CAR receptors, a synthetic T cell receptor that coordinates a suite of immune responses, and SynNotch, a synthetic receptor that can be programmed to activate just about any gene. In a mouse model of cancer, treatment with SNAP-CAR T cells shrunk tumors and greatly prolonged survival, an important proof-of-concept that sets the stage to test this approach in clinical trials in partnership with Coeptis Therapeutics, which has licensed the SNAP-CAR technology from Pitt. The discovery could extend into solid tumors and give more patients access to the game-changing results CAR T cell therapy has produced in certain blood cancers. With the addition of SNAP, the possibilities for customized therapies become almost endless.

 

 There is a pervasive sense of hopelessness in these non-small cell lung cancer (NSCLC) patients due to the current lack of treatment options, treatment failure due to drug resistance, and poor survival rates. Chemotherapy resistance is a major challenge in the treatment of NSCLC. Nuclear factor erythroid 2-related factor 2 (NRF2) may be at the root of this resistance. It is a master regulator of hundreds of other genes involved in numerous cytoprotective and metabolic pathways. Under normal physiological conditions, NRF2 protects cells from oxidative stress, toxic attacks and chemotherapy, keeping cells in homeostasis. Cancer cells hijack the NRF2 pathway and disrupt this function. When NRF2 becomes over-expressed and accumulates in the cell, the cell is able to fight off these toxic insults. This is really where the cancer becomes chemoresistant. Advanced patients have much higher levels of NRF2. However, in a paper published last month in Gene Therapy, a research team describes a new anti-cancer strategy that uses CRISPR-Cas9 to eliminate NRF2. The key finding of the study is that CRISPR-based disruption of NRF2 can lead to exon skipping. A dual sgRNA approach to target exon 4 resulted in the deletion of a 103-base-pair fragment, including exonic splice enhancer sequences. 

To restore dystrophin production and myocyte function in patients with Duchenne muscular dystrophy (DMD), several CRISPR strategies have been tried to correct some of the hundreds of thousands of documented mutations in the dystrophin gene (DMD). Among these strategies, researchers at the University of Texas Southwestern Medical Center in Dallas, Texas, have developed a novel CRISPR gene editing strategy for DMD therapy. This was tested in animal and human DMD models where the researchers deleted exon 51 in the DMD gene which disrupted the dystrophin reading frame and generated a premature stop codon in exon 52. The researchers were able to restore the reading frame with both basic and main editing by introducing exon skipping and reframing, respectively, through their "single-swap" ABE strategy, i.e., through editing a single letter in the splice acceptor or donor site can cause exon skipping. The researchers targeted the splice acceptor or splice donor site of exon 50 or 52 and the single nucleotide modification restored dystrophin expression in human cardiomyocytes and contractile function was normalized using master editing. However, one challenge is the high doses required to deliver expression throughout the body.

CAR T cell immunotherapies are more effective as a treatment for patients with leukemia or B cell lymphomas because once they are re-injected into the patient, they migrate and lodge in the bone marrow and other organs that make up the lymphatic system. However, for solid tumours, such as breast cancer, the CAR T cells have difficulty migrating to the tumour because of the microenvironment that surrounds it. Recently, a study conducted by researchers at the UNC Lineberger Comprehensive Cancer Center, shows that adding a small molecule to the CAR T cell-based treatment could stimulate the Th17 and Tc17 cells of the immune system to effectively attack solid tumours. To stimulate the accumulation of these Th17 and Tc17 cells in the vicinity of solid tumours, the research team discovered that the stimulator of interferon agonist (STING) genes, cGAMP, activates the human STING and is known to stimulate the human immune system. The various experiments showed that mice injected with cGAMP showed increased proliferation of T cells and these cells migrated to the tumour site. The end result was a significant decrease in tumour growth and improved survival.  

Researchers at Duke University have shown that a single systemic treatment using CRISPR genome editing technology can safely and stably correct a genetic disease -- Duchenne muscular dystrophy (DMD) -- for more than a year in mice, despite observed immune responses and alternative gene editing outcomes. The study appears online on February 18in the journal Nature Medicine.