Genetic Engineering Publications - GEG Tech top picks

CRISPR-based gene editing has become a mainstay of biological discovery, providing relatively rapid insight into the function of individual genes and targets for new therapies. However, the main challenge is that it is difficult to edit immune cells without changing their biology, hampering the ability to study immune cell behavior in all its complexity in a living organism. In an earlier study, researchers used CHimeric IMmune Editing (CHIME) to knock out a gene called Ptpn2, which has shown promise for cancer immunotherapy. The recent study published in Nature Immunology explores methods to increase the precision and versatility of CHIME, including simultaneous deletion of multiple genes and targeted application in specific cell types, also using CRISPR to disrupt genes in the edited cells once they were already back inside the animal. This research has successfully demonstrated the feasibility of various innovative approaches to genetic manipulation, offering new avenues for the study of immune gene function. 

Researchers have reported promising results in a Phase I/II trial involving 37 patients with relapsed or refractory B-cell malignancies who were treated with a cord blood-derived natural killer (NK) chimeric antigen receptor (CAR), a cell therapy targeting CD19. Results showed an overall response (OR) rate of 48.6% 100 days after treatment, with one-year progression-free survival (PFS) and overall survival (OS) rates of 32% and 68%, respectively. The trial reported an excellent safety profile, with no cases of cytokine release syndrome (CRS), neurotoxicity or graft-versus-host disease. Another key finding of the trial was the importance of allogeneic cord blood donor selection criteria in the manufacture of CAR NK cells. Cord blood units cryopreserved within 24 hours of collection and those with a low content of nucleated red blood cells were associated with significantly better results. CAR NK cells generated from these units resulted in a one-year PFS rate of 69% and an OS rate of 94%, compared with 5% and 48%, respectively, for units with higher nucleated red cell content or longer collection to cryopreservation times. 

Researchers explored the impact of dietary-derived trans-vaccenic acid (TVA) on effector cytotoxic T lymphocyte functions and anti-tumor immunity in in vivo settings using CRISPR. Overall, the results of the study showed that dietary trans-vaccenic acid enhances effector cytotoxic T lymphocyte activity and anti-tumor immunity in in vivo contexts. In contrast to intra-organism SCFA derived from intestinal microbes functioning as agonists of the G protein-coupled immunoregulatory receptor 43 (GPR43), extra-organism VAT reprogrammed CD8+ T lymphocytes by extrinsic regulation to inactivate GPR43. The results of the study contribute to a better understanding of the molecular links between nutrition and human pathophysiology, with implications for future research into the function of circulating nutrients in health and disease. Further research is needed to improve understanding of the effector pathways downstream of GPR43 and elucidate the underlying processes.

Until now, researchers have lacked the tools to create a targeted cell therapy approach that could work on all the different forms of blood and bone marrow cancers. A new solution could solve a major problem in immunotherapy, namely the inability to target surface markers present on both cancerous and healthy cells. In the study, published today in Science Translational Medicine, researchers used CAR T cells engineered to target CD45, a surface marker present on almost all blood cells, including almost all blood cancer cells. Since CD45 is also found on healthy blood cells, the research team used CRISPR base editing to develop a method called "epitope editing" to overcome the challenges of an anti-CD45 strategy, which would otherwise result in potentially lethal low blood counts and threatening side effects. The modified version of CD45 still functions, but differs sufficiently from normal CD45 for anti-CD45 CAR T cells not to recognize and attack it. This study lays the foundations for a more universal approach that could potentially extend CAR T cell therapy to all blood cancers. 

Immunotherapy is an essential component of cancer treatment. However, despite the success of immunotherapies for various types of cancer, only a few cancer patients have shown responses to current immune therapies and therapies can have adverse effects. Therefore, researchers have created intramuscular messenger ribonucleic acid (mRNA) lipid nanoparticle (LNP) vaccines as likely candidates for therapeutic cancer vaccines. RNA molecules can be encapsulated in LNPs. In addition, single-stranded mRNA can encode tumor vaccine neo-antigens, while small double-stranded interfering RNA can encode knockdown checkpoint inhibitors to adjust immune responses through RNA-induced activation of suppressed immune cells. Circular-type RNA can increase expression time, which benefits the generation of chimeric antigen receptors (CAR) in vivo and vaccine antigens. Targeted delivery of LNP in vivo would be essential for generating CAR-T macrophages and lymphocytes, in vivo. Based on the results of the study, the LNP mRNA vaccine platform could be used to develop next-generation personalized cancer vaccines. However, additional research is needed to further our understanding of cancer biology and improve vaccine design for faster clinical translation. 

Glaucoma is a neurodegenerative disease that causes vision loss and blindness due to a damaged optic nerve. Unfortunately, there is currently no cure. In a paper recently published in Communications Biology, researchers found that restoring mitochondrial homeostasis in diseased neurons can protect optic nerve cells from damage. The research team used induced pluripotent stem cells from glaucoma and non-glaucoma patients as well as clustered regularly spaced short palindromic repeats (CRISPRs) from human embryonic stem cells with glaucoma mutation. Using optic nerve stem cell differentiated retinal ganglion cells, electron microscopy and metabolic analysis, the researchers identified glaucomatous retinal ganglion cells with mitochondrial deficiency with a higher metabolic load on each mitochondrion, which leads to mitochondrial damage and degeneration. However, the process could be reversed by enhancing mitochondrial biogenesis with a pharmacological agent. The team showed that retinal ganglion cells are very efficient at degrading bad mitochondria, but at the same time produce more to maintain homeostasis.

CAR T cells have shown some success in the clinic in treating certain cancers, such as relapsed leukemia. However, CAR T cells have not been successful in delivering solid tumors, in part because of problems with immune cell activation. Adding a molecular anchor to the key protein used to recognize cancer in cellular immunotherapies can make treatments much more effective. The researchers found that immune cells with the anchored protein increased cancer killing, regardless of their cell type or the type of cancer targeted. The concept of molecular anchoring is thus a new design for improving chimeric antigen receptor (CAR) based immunotherapies. Anchored CARs have increased survival in animal models of several tumor types, including lung, bone and brain cancers. CARs have shown promise in the clinic, but have not yet achieved widespread success in all tumor types. The findings were published in Nature Biotechnology.

CAR T cell therapies are generally optimized for short-term cellular responses and may not allow for long-term systemic eradication of the tumor. To enable precise control of CAR T cell function over time, a first solution has been developed. Researchers have exploited recently developed synthetic Notch receptors to design enhanced CAR T cells with a second receptor. The second receptor can recognize a tumor antigen and then cause the T cell to release the cytokine interleukin-2, but when the CAR T cells are in direct contact with the tumor cells. In a mouse model, the approach enabled CAR T infiltration of solid pancreatic and melanoma tumors, resulting in substantial tumor eradication. In addition, a second solution was developed. Researchers developed a toolbox of 11 programmable synthetic transcription factors that could be activated on demand with timed administration of FDA-approved small-molecule inducers. Using these tools, the authors designed human immune cells that activate proliferation and antitumor activity on demand. The combination of the two technological advances presented will provide an unprecedented ability to precisely control the state of therapeutic cell populations not only at the time of injection but also as the immune response unfolds in the patient.

Many cancer stem cells possess an RAS gene that, when mutated, allows tissue stem cells to ignore normal environmental signals and control tissue growth. To better understand the intricacies of this interaction, researchers turned their attention to squamous cell carcinoma. The team began by inducing mutant HRAS (the most common RAS family member in skin cancers) in individual skin stem cells and monitoring the interaction of the cancer stem cells with the surrounding tissue. This observation raised the possibility that many cancer mutations do not fix the course of a disease but lock it in place, affirming a malignant progression already determined by aberrant crosstalk between a cancer stem cell and its microenvironment. By further studying how the cancer stem cell changed in the face of this new self-imposed malignant tumor microenvironment, the team realized that invasive cancer stem cells unexpectedly began expressing the leptin receptor, Lepr. The researchers used CRISPR technology to show that Lepr and leptin receptor signaling were critical for progression from benign to malignant. The team is now investigating ways to block leptin receptors in tumors because doing so could throw a molecular monkey wrench into the vicious loop and derail the cancer.

Breast cancer is the second most common cancer in American women, and triple-negative breast cancer (TNBC) is a more aggressive and deadly form of the disease that accounts for 10-15% of all breast tumors. Using a comprehensive, state-of-the-art genomic screening method known as CRISPR/CAS9 screening, scientists were able to identify a specific enzyme called UBA1 that proved to be an ideal therapeutic target. Using a new UBA inhibitor drug called TAK-243, they blocked the cellular function of UBA1 and effectively killed cancer cells in patient-derived breast tumors in mice. Previous research has shown that UBA1 inhibitors can have a positive impact on hematological cancers such as acute myeloid leukemia and chronic myeloid leukemia. This study, recently published in PNAS Nexus, is the first to suggest that UBA1 inhibitors may be effective in TNBC. TAK-243 was recently tested in early phase trials, paving the way for potential testing in TNBC patients. The researchers also determined that the c-MYC gene can be harnessed to cooperate with TAK-243 to initiate a cellular stress response and improve the drug's ability to combat TNBC. This supports the idea that TAK-243 may be effective in TNBC with high c-MYC expression, where c-MYC may serve as a biomarker for drug response.

Researchers examined two cohorts of patients who received CD19-directed CAR-T cells for non-Hodgkin's lymphoma or B-cell acute lymphoblastic leukemia. The researchers used the first cohort to retrospectively examine the impact of prior antibiotic use on CAR-T clinical outcomes. The second cohort had prospective baseline fecal samples collected before CAR-T cell infusion for subsequent assessment of the fecal microbiome. The investigators determined that the use of certain antibiotics including piperacillin/tazobactam, meropenem, and imipenem/cilastatin, negatively affected OS (HR = 1.71; 95% CI, 1.12-2.59). The investigators also noted a significantly increased risk of immune effector cell-associated neurotoxicity syndrome among the entire cohort evaluated for pre-CAR-T antibiotic use (P = 0.023). Stool samples from the prospective cohort demonstrated an altered fecal microbiome including lower alpha diversity, increased frequency of bacterial dominance, and altered bacterial composition compared with healthy controls. The results of this study suggest that the composition of the gut microbiome affects CAR-T outcomes, but the investigators cautioned that further prospective studies in larger patient populations are needed to confirm the associations.

Researchers have shown that a synthetic IL-9 receptor allows anti-cancer T cells to do their job without the need for chemo or radiation. T cells modified with the synthetic IL-9 receptor were potent against tumours in mice, as published in Nature. This group of researchers were interested in testing modified versions of the synthetic receptor that transmit other cytokine signals from the common gamma chain family: IL-4, -7, -9 and -21. Of the synthetic common gamma chain signals, the IL-9 signal was worth studying and unlike other cytokines, IL-9 signalling is not generally active in naturally ocurring T cells. The synthetic IL-9 signal gave the T cells a unique blend of stem cell and killer cell qualities that made them more robust in fighting tumours. In particular, the researchers targeted two types of difficult-to-treat cancer models in mice: pancreatic cancer and melanoma. They used T cells targeted to the cancer cells via the natural T cell receptor or a chimeric antigen receptor (CAR). In all cases, T cells engineered with synthetic IL-9 receptor signalling were superior and helped cure some tumours in mice when they could not do otherwise. 

A study published in Blood Advances reveals that magnetic resonance imaging (MRI) and lumbar puncture (LP) may not be systematically necessary in the diagnosis and management of severe cases of neurotoxicity linked to CAR T cell therapy. Instead, electroencephalogram (EEG), a non-invasive test, has proved useful in the management of these complications. The researchers examined the usefulness of these tests in 190 patients treated with CAR-T at Rennes University Hospital, where during treatment around 48% of patients developed immune effector cell-associated neurotoxicity syndrome (ICANS). The researchers assessed how the different tests affected patient treatment, such as how medications, e.g. antibiotics and anti-epileptic treatments, were prescribed based on abnormal results, and how these treatments altered patient outcomes. The results ultimately revealed that abnormal findings were more common in patients with more severe ICANS. MRI findings were often normal, and although LP and EEG often showed abnormalities, they were more common in more severe cases of ICANS. When it came to therapeutic decisions, MRI rarely led to changes, LP sometimes led to unnecessary treatments in cases of suspected infections, and EEG often resulted in adjustments to antiepileptic drugs.

In a recent study published in the journal Nature, researchers have compiled the available literature on natural killer (NK) cells, innate immune cells involved in the recognition and elimination of cells in distress, particularly virus-infected cells and tumors. They focus on reviewing current preclinical and clinical research in the field of NK therapies, primarily elucidating the role of NK cells in cancer immunity. They also explore the potential of bioengineering approaches to harness NK cells via the development of genetically modified NK cells, immune checkpoint inhibitors and cell engagement agents. The study reveals that, despite less than two decades of research in the field, NK cells are emerging as a safe, practical and potentially widely accessible means of clinical therapy, particularly antitumor. Although challenges exist in the adoption of NK cell therapies by conventional medicine, studies aimed at overcoming these challenges are already underway, bringing the future of clinical NK cell interventions closer than ever. 

Using CRISPR screening, the deubiquitinase ATXN3 was identified as a key regulator of PD-L1 transcription in tumor cells, a critical factor in tumor immune evasion. In this study, researchers transfected a targeted library of all 96 members of the mammalian deubiquitinase family into melanoma cells, then sorted the cells according to low and high PD-L1 expression to identify the regulators. ATXN3 was found to positively influence PD-L1 transcription, helping tumor cells to evade the immune system. This new insight represents a promising target for improving the efficacy of antitumor immunotherapy by blocking checkpoints, potentially transforming cancer treatment strategies. The study also highlights the broader role of ATXN3 in regulating tumor microenvironmental responses to hypoxia and inflammation, opening up new avenues for cancer research and treatment.

Solid tumors generate anti-immune signals that deactivate CAR T cells, making treatment less effective. To solve this problem, scientists have combined CAR T cells with cytokine injection, which can cause significant unintended toxicities. Researchers replaced the extracellular domain of various cytokine receptors with leucine zippers to create constitutively active receptors. CAR T cells expressing one of these chimeric cytokine receptors had superior antitumor activity against several types of cancer in cell lines and mouse models compared with conventional CAR T cells. Although chimeric cytokine receptors give a constant "on" signal to CAR T cells, they do not induce non-specific proliferation of CAR T cells. The system thus limits the effect of cytokine signaling to modified cells only, reduces the risk of cytokine-related toxicity, and provides a signal that these CAR T cells should function effectively in a suppressive tumor microenvironment. 

One of the challenges of CAR T cell therapy in solid tumors is a phenomenon known as T cell exhaustion. Previous studies have alluded to the inflammatory regulator Regnase-1 as a potential target to indirectly overcome the effects of T-cell exhaustion, as it can cause hyperinflammation when disrupted in T cells, reviving them to produce an antitumor response. The research team hypothesized that targeting the related but independent Roquin-1 regulator at the same time could boost responses further. The team used CRISPR-Cas9 gene editing to knock out Regnase-1 and Roquin-1 individually and together in healthy donor T cells with two different immune receptors that are currently being studied in Phase I clinical trials: the mesothelin-targeting M5 CAR (mesoCAR) and the NY-ESO-1-targeting 8F TCR (NYESO TCR). Following CRISPR editing, the T cells were expanded and infused into solid tumor mouse models, where the researchers observed that the double knockout resulted in at least a 10-fold increase in modified T cells compared to knocking down Regnase-1 alone, as well as increased anti-tumor immune activity and longevity of modified T cells. In some mice, this also led to an overproduction of lymphocytes, causing toxicity.

Pancreatic cancer is an incurable form of cancer and gene therapies are currently in clinical trials to treat this deadly disease. A comprehensive review of gene and cell biotherapies in development to combat pancreatic cancer is published in the peer-reviewed journal Human Gene Therapy. The article, "Pancreatic Cancer Cell and Gene Biotherapies: Past, Present and Future," by corresponding author Pierre Cordelier of the University of Toulouse, and co-authors describes ongoing gene therapy clinical trials. In addition to gene therapies, the authors discuss vaccines, chimeric antigen receptor (CAR) T-cell therapy, suicide genes and oncolytic viruses.

T-cell transfer therapies have not yet been successfully applied to solid tumors because T cells do not readily penetrate and persist in solid tumor masses for long periods of time, and because their activity is attenuated by an immunosuppressive tumor microenvironment. One way to overcome these limitations could be to couple T cell transfer therapies with cytokine therapy. However, a serious drawback of this approach is the significant side effects resulting from cytokines circulating freely in the body, leading to toxicity and potentially fatal inflammatory syndromes. Now, researchers have developed a nanotechnology-based solution to these problems. The method uses an unnatural sugar that is absorbed and embedded in the outer coating of T cells, which can then be used to anchor cytokines. The concentrated cytokines improve T-cell function locally without producing unwanted systemic side effects. In mice with melanoma, the approach also stimulated the host immune system against tumor cells, which inhibited tumor growth. As an adjunct to CAR-T cell therapy, it resulted in complete regression of lymphoma tumors at otherwise non-curative cell doses.

Researchers have bolstered the power of chimeric antigen receptor (CAR)-T cancer therapies, which use genetically altered T cells to seek out tumours and mark them for destruction. Now scientists have further engineered the cells to contain switches that allow control over when and where the cells are active. This helps them to infiltrate tumours and dodge immune-suppressing defences.

In most cases, the number of naturally occurring cancer-targeting T cells will be far too low to trigger an immune response capable of eradicating a patient's tumor(s). PACT Pharma is a privately held biopharmaceutical company developing personalized, neo-antigen-specific T-cell receptor (TCR)-T therapies to treat a range of solid tumors.  Patient T cells are isolated and CRISPR-modified by electroporation with Cas9 protein, guide RNAs to knock out endogenous TCR genes (TCRα ( TRAC) and TCRβ ( TRBC)) and an HR template plasmid encoding the transgenic neoTCR. The results of a phase 1 clinical trial that were published in Nature. The researchers report that CRISPR-modified T cells were preferentially directed to the tumor and could be recovered from post-infusion biopsies in all patients for whom biopsies were available. They also note that CRISPR-edited T cells frequently accounted for the top 2-20% of immune cells in the tumor, and a reduction in tumor size was observed in some lesions in a single lung cancer patient.

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.

Currently, too few CAR T cells become memory cells that persist and create more T cells in the long term. However, a group of researchers recently showed that the distribution of the c-Myc protein in a parental T cell may be important for this process and published this work in the journal Nature. The researchers knew that a daughter cell with more c-Myc became an effector cell. In this study, the team found that the protein complex cBAF (canonical Brg1/Brg-associated factor) interacted with c-Myc. Daughter cells with high concentrations of cBAF and c-Myc became effector T cells. The cBAF binds certain regions of chromatin, proteins on DNA. The discovery suggests that it can guide the fate of cells, what type of T cells they become, by controlling the expression of effector cell-related genes. The distribution of cBAF occurs in the first activated T cell that begins the adaptive immune response; therefore, the researchers realized that cell fate is decided early in the immune response. The researchers used the molecular information they discovered. They applied a cBAF inhibitor during CAR T cell activation to generate more memory T cells. In a preclinical model, T cells treated with an inhibitor-controlled tumor growth better than untreated cells. The treated cells also survived longer and in greater numbers.