Genetic Engineering Publications - GEG Tech top picks

In a review, researchers detail the development history of Principal Editing (PE), the latest evolution of CRISPR-Cas-based technologies. PE was proposed by a team of researchers in 2019, which is characterized by the absence of double-strand breaks (DSBs) or homology sequence patterns with variable application scenarios, including point mutations as well as insertions or deletions. The PE system consists of two parts: the master editors (PEs) and the master editing guide RNA (pegRNA). This PE system has developed and progressed rapidly over the last four years, with versatile advances in its architecture to increase editing efficiency, targeting and specificity, including a new pegRNA design, PE modification and improved delivery. Moreover, despite its relatively recent inception, PE has been widely applied to correct pathological mutations associated with genetic diseases, both in vitro and in vivo , presenting great potential for advancing the field of gene therapy from bench to bedside.

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.

Researchers are improving non-invasive treatment options for degenerative disc disease, a condition that affects 3 million adults each year in the U.S. Stem cell therapy has been a viable field in regenerative technologies for many pathologies for several years. However, the degenerated intervertebral disc provides a hostile environment that is detrimental to stem cell survival, resulting in limited clinical success of stem cell therapy for the disc. Previous research has shown that stem cell-derived electrical vehicles contain many therapeutically beneficial proteins, lipids and nucleic acids and carry much of the regenerative potential of stem cells. However, by using CRISPR in mesenchymal stem cells, researchers have added to the growing field of regenerative medicine the process of producing cell-based therapies to alleviate pain and lack of mobility. In their study, the researchers target TSG6, an essential stem cell marker known to be linked to the regenerative and anti-inflammatory properties of these stem cells.  Their hypothesis is that if they CRISPR-activate TSG6 in stem cells, they will not only increase TSG6 protein levels in the extracellular vesicle cargo, but potentially amplify the stem cells' anti-inflammatory and regenerative properties. 

Fabry disease is caused by a genetic deficiency of α-galactosidase A (GLA), leading to the accumulation of its substrates such as globotriaosylceramide and globotriaosylsphingosine. Researchers have therefore developed a modified enzyme, modified α-N-acetylgalactosaminidase (mNAGA), to cure Fabry disease by changing the substrate specificity of NAGA to that of GLA. In this study, researchers tested whether genome-editing transplantation of mNAGA-secreting induced pluripotent stem cells (iPS) cells could deliver GLA activity in vivo. They therefore generated mNAGA-secreting iPS cells by TALEN-mediated knock-in at the AAVS1 site, a refuge locus. Furthermore, to exclude possible immunogenic reactions caused by endogenous GLA from iPS cells in patients, they disrupted the GLA gene by CRISPR-Cas9. When cardiomyocytes and fibroblasts from the Fabry model without GLA activity were co-cultured with mNAGA-secreting iPS cells, GLA activity was restored by mNAGA-expressing cells in vitro. Next, they transplanted the mNAGA-secreting iPS cells into the testes of mouse models of Fabry disease. After 7 or 8 weeks, GLA activity in the liver was significantly improved, although no recovery of activity was observed in the heart, kidneys or blood plasma.

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.

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.

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.

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.

CAR T cells currently in clinical use are designed to recognize cancer cells directly and have successfully treated several blood cancers. But there have been challenges that prevent their effective use in many solid tumors. Most solid tumors are heavily infiltrated by a type of immune cell called macrophages. Macrophages help tumors grow by blocking the entry of T cells into the tumor tissue, which prevents CAR T cells and the patient's own T cells from destroying the cancer cells. To address this immune suppression at the source, the researchers engineered T cells to make a chimeric antigen receptor that recognizes a molecule on the surface of macrophages. When these CAR T cells encountered a tumor macrophage, the CAR T cell became activated and killed the tumor macrophage. Treating mice with ovarian, lung and pancreatic tumors with these macrophage-targeting CAR T cells reduced the number of tumor macrophages, shrank the tumors and prolonged their survival. The destruction of tumor macrophages allowed the mice's own T cells to access and kill the cancer cells. The researchers further demonstrated that this anti-tumor immunity was induced by the release of the cytokine interferon-gamma by CAR T cells.

A new study from Tel Aviv University proposes a new and unique AIDS treatment that could be developed into a vaccine or a one-time treatment for HIV patients. The study examined the engineering of B-type white blood cells in the patient's body to secrete anti-HIV antibodies in response to the virus. The technique developed in his laboratory uses B-white blood cells that would be genetically modified inside the patient's body to secrete neutralizing antibodies against the HIV virus. The gene editing was done with a CRISPR system. The researchers are able to engineer the B cells inside the patient's body using two viral vectors from the AAV family, one encodes the desired antibody and the second encodes the CRISPR system. When CRISPR cuts the desired site in the genome of the B cells it directs the introduction of the desired gene: the gene coding for the antibody against the HIV virus. On the basis of this study, we can hope that in the next few years we will be able to produce a drug against AIDS in this way, but also against other infectious diseases, for certain types of cancer caused by a virus, such as cervical cancer. 

The CAR T cell-based therapy Yescarta (axicabtagene ciloleucel) and manufactured by Kite Pharmaceuticals, was first approved by the U.S. Food and Drug Administration in 2017 as a third-line treatment for adults with large B-cell lymphoma (LBCL), i.e., for those who had already undergone two cycles of failed treatment. The ZUMA-7 clinical trial was therefore set up to determine whether a single infusion of Yescarta is superior to the existing and long-standing standard of second-line care, which is a stem cell transplant after high-dose chemotherapy to kill the lymphoma. According to the results of a clinical trial published December 11th and presented the same day at the American Society of Hematology annual meeting, this treatment is significantly more effective than the current standard of care in treating people with large B-cell lymphoma who relapse after first-line treatment. About 40% of people with LBCL need such second-line treatment, either because their cancer recurs or does not respond adequately to first-line treatment.

CAR T cells have proven to be a powerful weapon against blood cancers, but against solid tumors they are much less effective, in part because of a process called T cell exhaustion. However, Penn Medicine researchers have uncovered key molecular details of this exhaustion process that point to a specific strategy to overcome it. In this study, the scientists developed a labmodel  allowing them to thoroughly study the exhaustion process of CAR T cells designed to attack pancreatic tumors. They observed that the process of T cell exhaustion in the model closely resembled that observed in patient T cells. The model also revealed new facets of the exhaustion process, including the role of 2 genetic regulators of exhaustion, ID3 and SOX4, whose silencing allowed CAR T cells to retain much of their effectiveness against tumor cells. The scientists observed that CAR T cell exhaustion was accompanied by increases in the levels of two proteins, ID3 and SOX4, which function as master switches for large sets of genes in immune cells. Silencing these apparent T-cell exhaustion switches allowed the depleted CAR T cells to retain much of their antitumor efficacy even after prolonged exposure to tumor cells.

The study thus points to a specific strategy - inhibition of ID3 and/or SOX4 - that could help CAR T cells function better against solid tumors.

According to the results of the phase 1 trial published in Cancer Discovery, the combination of CAR T cell therapy and pembrolizumab, an anti-PD-1 therapy, appeared safe and feasible for patients with malignant pleural mesothelioma. 83% of patients who received the regimen remained alive 1 year after CAR-T infusion. The investigators reported no cases of high-grade cytokine release syndrome or neurotoxicity. This study was done with the use of a dose escalation of autologous CAR T cells that target the mesothelin protein on the surface of cancer cells and express an iCaspase-9 gene that serves as a "safety switch" to shut down the activity of the engineered T cells.

The investigators chose a locoregional CAR-T dosing strategy because this type of cancer does not typically metastasize outside the chest cavity.  A Phase 2 study using a fixed dose of mesothelin-directed CAR T cells followed by pembrolizumab is already underway. Four patients have been treated to date.

Traditional CAR-T cells, while effective against liquid cancers, face challenges in solid tumors: the cells wear out and ultimately fail to destroy the cancer completely. Ground-breaking research is providing an innovative approach to this challenge. Researchers are introducing CAR-T cells that excrete the IL-10 molecule. In other words, the cell has been designed to produce its own "drug" to stay healthy in the tumor's hostile environment. In the laboratory, this innovative CAR-T therapy systematically eradicated cancerous tumors in mouse models. What's more, in ongoing clinical trials, eleven patients have appeared to achieve complete remission with this treatment, representing a 100% success rate to date. Notably, the evidence from the laboratory study suggests the long-term efficacy of the therapy, and indicates that its manufacture could be both faster and more cost-effective than current methods 

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. 

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.

Researchers report, in Nature Medicine, the interim results of a first-in-man phase 1 clinical trial evaluating the safety, antitumor activity and immunological characteristics of a genetically engineered natural killer (NKT) cell immunotherapy for neuroblastoma, a childhood tumor that most commonly arises in the adrenal gland. NKT cells were engineered to express a GD2-specific CAR, which enables immune cells to target a molecule found on the surface of neuroblastoma cells, and interleukin-15 a natural protein that supports NKT cell survival. Based on results obtained in 12 patients with recurrent stage 4 neuroblastoma resistant to other therapies, the researchers found that the treatment was safe for all 12 patients on four doses. No dose-limiting toxicities were reported. A further discovery revealed a regulatory gene in NKT cells that could have an impact on treatment efficacy. Leveraging the multiomics platform of key collaborator Immunai, Inc. the researchers discovered that up-regulation of the anti-proliferation factor 1 gene BTG in CAR NKT-infused cells indicates cell exhaustion and limits the functional activity of CAR NKT cells. Conversely, artificially reducing BTG1 expression in CAR NKT cells enhanced their therapeutic activity against neuroblastoma in a mouse model

A new study shows that some effective cancer treatments, such as CAR-T cells, significantly improve quality of life six months after receiving therapy. To conduct the study, researchers recruited 103 patients aged 23 to 90 years with a diagnosis of blood cancer from April 2019 to November 2021. The researchers administered self-reported questionnaires measuring quality of life variables at time intervals including before CAR-T cell infusion and one week, one month, three months, and six months after CAR-T cell infusion. Quality of life was measured using a 27-item questionnaire known as the General Cancer Therapy Functional Assessment, which is composed of four different subscales (physical, functional, emotional, and social). Psychological distress was measured using the Hospital Anxiety and Depression Scale. Finally, major depressive symptoms were measured using the PHQ-9, and symptoms of posttraumatic stress disorder were measured using the Posttraumatic Stress Checklist. While most study participants eventually experienced an improvement in quality of life, approximately 20% of patients experienced persistent physical and psychological symptoms, which at times interfered with their quality of life.

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.

Crystal Mackall remembers her scepticism the first time she heard a talk about a way to engineer T cells to recognize and kill cancer. Sitting in the audience at a 1996 meeting in Germany, the paediatric oncologist turned to the person next to her and said: “No way. That’s too crazy.”

Today, things are different. “I’ve been humbled,” says Mackall, who now works at Stanford University in California developing such cells to treat brain tumours.

Researchers have used CRISPR/Cas9 technology to engineer donor T cells to treat critically ill children with resistant leukemia who had otherwise exhausted all available therapies. The Phase I trial, published in Science Translational Medicine , is the first use of CRISPR-edited "universal" cells in humans and represents a significant advance in the use of genetically engineered cells for cancer treatment. In the trial, the research team constructed and applied a new generation of genome-edited "universal" T cells, which builds on previous work that used older, less precise technology. The T cells were modified using CRISPR. In addition, the piece of genetic code allows the T cells to express a CAR receptor that can recognize a marker on the surface of cancerous B cells and then destroy them. The T cells were then genetically modified with CRISPR so that they could be used "off the shelf" without the need for a donor match. In specialist clean rooms, researchers manufactured their banks of donor CAR T-cells using a single disabled virus to transfer both the CAR and a CRISPR guidance system, and then applied cutting-edge mRNA technology to activate the gene editing steps.

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.

Researchers at Seidman Cancer Center and Case Western Reserve University Hospitals have developed a new approach to CAR T cell therapy for B-cell cancers that triples targeted antigens on cancer cells. This approach promises to significantly reduce the potential for antigen escape currently found in CAR T therapies that target only CD19. The novel B-cell activating factor (BAFF) CAR T product developed here specifically binds to each of three receptors instead of one - BAFF-R, BCMA and TACI, providing more therapeutic options. At least two of these three receptors are found in almost all B-cell cancers, with some cancers expressing all three. Experimental results reported in Nature Communications show that BAFF CAR T is effective in killing several B-cell cancers. In addition, studies show robust in vitro and in vivo cytotoxicity exerted by CAR T BAFFs against mantle cell lymphoma, multiple myeloma, and mouse xenograft models of acute lymphoblastic leukemia. An Investigational New Drug application with the U.S. Food and Drug Administration will be filed in the coming weeks with Luminary Therapeutics and the team plans to initiate a clinical trial of BAFF CAR T therapy in patients with non-Hodgkin's lymphoma within the next few months. 

Multiple myeloma is a complex and incurable type of blood plasma cancer that often requires multiple treatments as the disease progresses and becomes resistant to previous treatments, often resulting in chronic disease with periods of acute illness. CAR-T cells are used to target a protein known as B-cell maturation antigen

(BCMA) that is commonly found in multiple myeloma. This therapy has shown impressive response rates in people with particularly complex and treatment-resistant multiple myeloma. However, according to scientists at Icahn Mount Sinai, 3 months after injection of CAR-T cells, 1 patient began to show progressive neurological features of Parkinson's-like symptoms. The patient died as a result of the treatment. Researchers found BCMA in the basal ganglia of his brain and scarring in that area. This serious side effect could be due to the therapy targeting BCMA in the brain. Thus, this study shows that BCMA-targeted CAR-T cell therapy can cross the blood-brain barrier in at least a subset of patients and can cause progressive neurocognitive and movement impairment. In addition, the scientists also found BCMA expression in the brains of healthy individuals. CAR-T cell therapies, while effective in multiple myeloma, warrant close monitoring for neurotoxicity.