Genetic Engineering in the Press by GEG

CAR-T cells have shown clinical efficacy in blood cancers, but have not been as effective in solid tumors. This variation in efficacy is partly due to the fact that tumors promote T-cell depletion, in which the cells are less effective at actively killing cancer. The researchers therefore created a map for the domain of T-cell differentiation and depletion in tumors, based on high-dimensional loss-of-function genetic screening. The researchers thus mapped the transcription factors expressed in tumor-infiltrating T cells. This map will provide a guide that future researchers can refer to and use to identify ways of improving T-cell-based immunotherapies. The researchers achieved this feat using single-cell CRISPR-Cas9 screening, a gene-editing technology that analyzes the gene expression profiling of individual cells after selectively eliminating transcription factors in a comprehensive screen. By examining the gene expression pattern of an individual T lymphocyte, the researchers were able to compare them to determine which inactivated transcription factors most affected T cell differentiation and anti-cancer activity. The same approach could be more widely applicable to increase knowledge in a number of contexts.

One of the difficulties that has hampered the success of CAR T cells in brain and solid tumors is the difficulty of identifying a good target for these cancers. However, researchers have discovered that GRP78 is an excellent target for CAR-T cells. The researchers then created CAR T cells targeted on GRP78 that successfully killed many types of cancer in cell and mouse models, although with significant variations. The researchers expected that higher levels of GRP78 would make it easier for CAR T cells to localize and destroy cancer. However, this was not the case. The scientists found no relationship between the amount of GRP78 and the ability of CAR T cells to kill cancer. In fact, the resistant tumor cell types modified the CAR T cells. Tumor cells induced GRP78-targeted CAR T cells to express GRP78 on the CAR T cell surface. The more GRP78 on the T cells, the less active they became, reducing their anti-cancer activity. Moreover, CAR T cells that remained active targeted and killed their counterparts expressing GRP78 on their surface. The protein remains an interesting target, given its presence on many difficult-to-treat tumor types. However, scientists will need to broaden their understanding of this new interaction with T cells to create viable immunotherapies. 

No organism on earth is immune to threats, including bacteria. Phages are among their most formidable enemies, infiltrating their cells to replicate and take over. Bacteria have developed a range of strategies to counter these infections, but how they first detect an invader in their midst has long been a mystery. Researchers pioneering the study of bacterial defense systems, principally CRISPR-Cas, have focused on the immune response system of Staphylococcus schleiferi. This led them to hypothesize that these sensitive phages produce, during infection, an element that triggers activation of the cyclic oligonucleotide-based antiphage signaling system (CBASS). Next, the researchers tested various molecules produced by the bacteria or virus, including DNA, RNA and proteins. The experiment revealed that only RNA produced during a phage infection was capable of triggering an immune response. Thus, CBASS detects a specific RNA structure. The researchers then invented the newly-identified hairpin-shaped cabRNA molecule for CBASS-activating bacteriophage RNA. The molecule binds to a cyclase surface, triggering the production of a messenger molecule called cGAMP which activates the CBASS immune response. These discoveries could one day help counter the threat of antibiotic resistance.

Researchers have explored the effects of nicotinamide mononucleotide (NMN) on HIV-1 infection in primary CD4+ T cells. They found that NMN treatment increased intracellular levels of nicotinamide adenine dinucleotide (NAD) and suppressed HIV-1 replication, as evidenced by reduced p24 viral protein production in infected cells. Despite these findings, NMN did not significantly affect the early stages of the HIV-1 life cycle, such as viral entry, reverse transcription, integration and transcription. The researchers also examined the effects of NMN on CD25 + CD4 + T cells and HIV-1 replication. They found that NMN treatment reduced the frequency of CD25 + cells and human leukocyte antigen - DR positive (HLA-DR + ) and significantly suppressed intracellular p24 in CD25 + CD4 + T cells. This suggests that NMN may affect the proliferation of infected cells. In addition, NMN was found to modulate CD25 expression in specific subsets of CD4 + T cells and reduce proliferation of primary p24 + CD4 + T cells via down-regulation of CD25. Using an in vivo HIV-1-infected humanized mouse model, the researchers found that combined NMN and CART treatment significantly improved CD4+ T cell reconstitution compared with CART alone.

Scientists have developed a new method for engineering more potent immune cells that can potentially be used for "off-the-shelf" cell therapy to treat challenging cancers. The team focused on gamma delta T cells, an immune cell known for its ability to target a wide range of cancers, including solid tumors, without causing graft-versus-host disease, a common complication in allogeneic cell therapies. Although gamma delta T-cell therapies have been studied before, they have had limited clinical success due to donor variability, short-lived persistence and the ability of cancer cells to evade or avoid the body's immune response. However, the researchers found that donor gamma delta T cells with high expressions of a CD16 surface marker had a greater ability to kill cancer cells. The team of scientists was able to efficiently produce the most potent cells in large quantities, which they then tested on two different preclinical models of ovarian cancer. They found that the cells were capable of attacking tumors and remained in the models for a long time, enabling them to continue their anti-tumor effects. In addition, there was no evidence of complications such as graft-versus-host disease.

By 2022, over 1,400 different types of T-cell therapy were in development, seven of which have been approved by the FDA for various cancer treatments. Use of the therapy, called chimeric antigen receptor T cell (CAR-T), is limited, however, due to the cost and time required to grow the T cells. Each infusion treatment for a cancer patient requires up to 250 million cells. The manufacturing demand for this growing number of therapies is not being met, so there is a gap to be filled in terms of biomanufacturing solutions. Researchers have developed a bioreactor the size of a mini-refrigerator, capable of manufacturing cells called T cells at 95% of maximum growth rate, some 30% faster than current technologies. The bioreactor uses centrifugal force to act on the growing cells while they are suspended in a dense cloud and continuously bathed by the incoming flow of nutrient-containing medium. The researchers developed it using T cells from cattle, and expect it to work in the same way on human cells.

Pancreatic ductal adenocarcinoma (PDAC) cells have a high proliferative capacity and a high demand for nutrients, i.e. amino acids, sugars and lipids. Amino acids are particularly associated with cancer growth. The aim of this study was to investigate SLC38A5 (SN2/SNAT5), a sodium-coupled neutral amino acid transporter, for its connection to PDAC. SLC38A5 transports glutamine, asparagine, methionine, glycine and serine. The study revealed that SLC38A5 is significantly up-regulated in PDAC. This overexpression of SLC38A5 at mRNA level results in poor survival in PDAC patients. A CRISPR/Cas9-mediated inactivation experiment revealed the tumor-promoting role of SLC38A5. Genomic and metabolomic studies indicated that deletion of SLC38A5 leads to a decrease in many SLC38A5 amino acid substrates and the inactivation of a fundamental mitochondrial process, namely OXPHOS. Furthermore, deletion of SLC38A5 also causes inhibition of the mTORC1 signaling pathway, as well as mitochondrial glycolysis and respiration. Given the experimental results of this study, SLC38A5 was identified as a key promoter of PDAC. Consequently, it could be used as a potential therapeutic target to develop new treatments for PDAC. 

Using laboratory-grown cells from genetically modified humans and mice, researchers claim to have evidence that modifying a specific protein called Bourneville tuberous sclerosis protein 2 (TSC2) in CD8+ T lymphocytes can make these cells more robust, potentially opening the door to better use of T cells in people's immune systems to fight cancer. The TSC2 protein can normally activate or block a molecular pathway that regulates T cells. Their experiments show that the introduction of a mutation in the TSC2 gene can lead to increased or decreased activation of this T cell regulatory pathway when T cells actively respond to immune challenges such as a cancer antigen. This discovery opens up the possibility of improving CAR-T cell-based therapies, in which T cells are genetically modified to better recognize a particular cancer. The scientists also discovered that T cells containing the TSC2 mutation could develop in large numbers during the initial immune response, but could also persist over the long term.

In a recent study, researchers explored the therapeutic impact of Thiostrepton (TST) on experimental colitis by examining its effects on colonic inflammation and determining its targets through a variety of biochemical approaches. In particular, CRISPR-Cas9 technology was used to generate cell lines. In the study, the therapeutic and prophylactic efficacy of TCT on dextran sulfate sodium (DSS)-induced colitis in mice was investigated, revealing a significant attenuation of disease phenotypes, evident by attenuated weight loss, reduced disease activity index scores and attenuated colon shortening and histological severity. Closer examination of inflammatory cytokine production revealed that TST decreased their production levels. The frequency of interleukin-17A-producing cells in the colon of DSS-exposed mice was significantly reduced after TST treatment, mainly affecting Gamma delta T cells in inflamed colons. Experiments in mice suggest that the protective efficacy of TST depends on the presence of RORγt, marking RORγt-expressing IL-17A-producing cells as selective targets of TST in DSS-induced colitis. Thus, TST appears as a potential immunosuppressant to treat autoimmune diseases such as inflammatory bowel diseases.

Glioblastomas are the most aggressive of all brain tumors. They spread diffusely throughout the brain and are difficult to remove completely by surgery. Chemotherapy or radiotherapy also often have limited efficacy. To find new, more effective treatment options for those affected, researchers are building on a current study into the relatively new concept of "transgenic T-cell receptor cells". Brain tumor patients were first inoculated with an antigenic fragment of the NLGN4X protein (Neuroligin4X). This protein is found in large quantities in glioblastoma cells, but is virtually undetectable in healthy brain tissue. The researchers then isolated activated T cells that recognized NLGN4X from the blood of vaccinated individuals. The researchers then demonstrated that NLGN4X-specific T cells were able to kill brain tumour cells in the culture dish. Brain tumor-bearing mice treated with NLGN4X-specific transgenic human T cells had a treatment response of over 40%. The tumors shrank and the animals survived longer than their untreated counterparts.

Six CAR T cell therapies have received FDA approval, and several more are in the pipeline. However, these therapies carry serious and potentially fatal side effects, namely cytokine release syndrome (CRS) and neurotoxicity. These drawbacks manifest themselves in a range of symptoms, from high fever and vomiting to multivisceral failure and patient death, posing significant challenges for wider clinical application. Now, a research team has found a solution that could help CAR T therapies reach their full potential while minimizing serious side effects. Their findings are published in the journal Nature Materials. Undesirable interactions between CAR T and immune cells called macrophages lead to macrophage overactivation, resulting in the release of toxic cytokines leading to CRS and neurotoxicity. Controlling CAR T-macrophage interactions in vivo is challenging. Thus, the study introduces a materials engineering-based strategy of incorporating a sugar molecule into the surface of CAR T cells becoming "pegylated CAR T cells". These sugars are then used as a reactive handle to create a biomaterial coating around these cells directly in the body, which acts as an "armor", preventing dangerous interactions with macrophages.

Triple-negative breast cancer (TNBC) affects 10-15% of breast cancer patients in the USA. TNBC is called "triple-negative" because the cancer lacks the estrogen receptor, the progesterone receptor and the HER2 receptor. The absence of any of these receptors makes treatment of TNBC quite difficult, and TNBC patients have limited treatment options. All these factors contribute to TNBC patients suffering a poorer prognosis. Researchers have studied and demonstrated the efficacy of a dual-effect concept: silencing the ACSS2 gene alters TNBC metabolism while simultaneously boosting the immune system's ability to fight it. ACSS2 regulates acetate, a nutrient that cancer cells and TNBC cells use to grow and spread. The researchers used two methods to deactivate ACSS2: CRISPR-Cas9 gene editing and the compound VY-3-135, a potent ACSS2 inhibitor. The researchers found that targeting ACSS2 in this preclinical study not only hindered the ability of this aggressive cancer to metabolize acetate and grow, but also prompted the immune system to recognize and attack the cancer.

Yesterday, the U.S. Food and Drug Administration (FDA) surprised the CAR-T community by announcing that it’s investigating blood cancer cases apparently caused by CAR-T therapy. Until now, such a hazard for the approved treatments hadn’t been recognized. “This is very unexpected,” says CAR-T researcher Michel Sadelain of the Memorial Sloan Kettering Cancer Center (MSK).

Four children born deaf regained almost normal hearing a few weeks after receiving gene therapy, announced a Chinese medical team from Fudan University in Shanghai.

The tendency of CAR T cells to lose their function over time, a phenomenon known as T-cell exhaustion, has been a major obstacle to treatment. Even when CAR T cells are effective in the short term, cancer often returns. This problem partly explains why CAR T cell therapy has not worked as well against solid tumors as it has against blood cancers. However, the researchers noted that disruption of the SUV39H1 gene has a knock-on effect: it restores the expression of several genes that help maintain T cell longevity. The researchers used the CRISPR/Cas9 gene-editing tool to modify SUV39H1 in human CAR T cells. They placed these modified CAR T cells in mice that had been implanted with either human leukemia cells or prostate cancer cells. For both cancers, the CAR T cells were able to maintain their function without depleting, leading to tumor elimination. In contrast, mice with unedited CAR T cells did not survive the cancer. There did not appear to be any serious side effects in the mice, although researchers will need to confirm the safety of this approach in humans. This new approach also requires fewer CAR T cells, and could therefore broaden the pool of patients likely to be eligible for this treatment

CAR-T cell therapy is a last hope for many patients with cancer of the blood, bone marrow or lymph nodes when other treatments such as chemotherapy fail. A limiting factor of this otherwise highly effective and safe therapy is that the cells used in the process rapidly reach a state of exhaustion. In a recent study, researchers produced four types of CAR-T cells, each expressing a CAR with each of the four signaling subunits (zeta, gamma, delta and epsilon), and tested them in a mouse model of leukemia. Surprisingly, the zeta chain, the domain used in clinically-applied CAR-T cells, showed a lower anti-tumor effect than the other three domains. The latter were significantly better at eliminating cancer cells from the leukemia model. The researchers explain this result by the fact that, although the zeta chain transmits a powerful activation signal to the cell, it is also rapidly depleted. In contrast, the delta chain, which showed the greatest efficacy in the present study, triggers an inhibitory signal parallel to T cell activation, enabling the immune cell to function at its optimal speed.

Multiple myeloma, a cancer of the plasma cells in the bone marrow, is the second most common blood cancer in the United States, with a five-year survival rate of 56%. Among black and Hispanic populations, it is the most common blood cancer. Non-Hispanic black individuals are twice as likely to develop multiple myeloma as their white counterparts. Although several treatments exist, the disease remains incurable and most patients who achieve remission end up relapsing. In 2021, idecabtagene vicleucel became the first CAR-T cell therapy to receive U.S. Food and Drug Administration approval to treat relapsed and refractory multiple myeloma, a version of blood cancer that has not responded to treatment. A recent study shows that multiple myeloma patients treated with idecabtagene vicleucel showed no difference in overall survival outcomes, regardless of race or ethnicity.

Synthetic biologists have created a new approach to attacking tumors. They have engineered tumor-colonizing bacteria (probiotics) to produce synthetic targets in tumors that direct CAR-T cells to destroy newly highlighted cancer cells. The researchers have essentially created a universal CAR-T cell that attacks a universal antigen, by programming tumor-seeking bacteria to paint solid tumors with a synthetic marker that CAR-T cells can recognize. They expect that, with further improvements, this platform will enable treatment for any type of solid tumor without the need to identify a specific tumor antigen, thus avoiding the need to generate a customized CAR-T cell product for each type of cancer and each patient. The researchers continue to refine their work and hope to launch clinical trials to fully evaluate the platform's safety and efficacy in human patients.

Regeneron Pharmaceuticals Inc. and Intellia Therapeutics Inc. have announced an expanded research collaboration aimed at developing further CRISPR-based gene-editing therapies focused on neurological and muscular diseases. According to an Intellia press release, the expanded partnership aims to combine Regeneron's proprietary adeno-associated viral vectors and antibody-targeted delivery systems with Intellia's proprietary Nme2 CRISPR/Cas9 systems tailored to the delivery of viral vectors that modify targeted genes. Under the terms of a recently expanded agreement, the companies will initially pursue two non-hepatic targets in vivo, with Intellia leading the design of the editing methodology while Regeneron will lead the design of the targeted viral vector delivery approach. According to the press release, the agreement further stipulates that each partner may lead the potential development and commercialization of product candidates for a target, while the company not leading development and commercialization may enter into a co-development and co-commercialization agreement for that target.

Three base-editing therapeutic candidates are currently involved in clinical trials: 

• Verve Therapeutics is developing VERVE-101 as a single-dose base-editing therapy for heterozygous familial hypercholesterolemia. VERVE-101 is currently being evaluated in the Heart-1 Phase 1 clinical trial in the UK and New Zealand. 
• The Great Ormond Street Hospital for Children NHS Foundation Trust in the UK is sponsoring a Phase 1 trial for BE CAR-7, a proprietary CAR T cell therapy designed to treat various T cell cancers such as acute cellular lymphocytic leukemia. 
• Beam Therapeutics announced that the U.S. FDA had approved BEAM-101 for clinical evaluation as a treatment for sickle cell disease. The company announced in a press release last month that the first patient had received the company's second clinical-stage basic editing therapeutic candidate, BEAM-201. BEAM-201 is a therapy in development for the treatment of relapsed or refractory T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

There are different types of CRISPR system, and the one on which researchers have focused to describe the three-dimensional structure in detail is called CRISPR-Cas13bt3. What's unique about it is that it's very small. Usually, these types of molecules contain around 1,200 amino acids, whereas this one only contains around 700. Smaller size is a plus, as it allows better access and delivery to target editing sites. To obtain the three-dimensional structure, the researchers used a cryoelectron microscope to map the structure of the CRISPR system, placing the molecule on a thin layer of ice and projecting an electron beam through it to generate data which was then processed into a detailed three-dimensional model. CRISPR-Cas13bt3 is totally different from CRISPR-Cas9: the scissors are already there, but they have to hook onto the RNA strand at the right target site. To do this, it uses a binding element on those two unique loops that link the different parts of the protein together. The researchers then used their findings to refine the tool to increase its precision, and tested its activity and specificity in living cells. They found that in cell cultures, these systems were able to target much more easily.

Among the immunotherapies available, the use of CAR-T cells is proving extremely effective against certain blood cancers, but only in half of patients. One of the main reasons for this is the premature dysfunction of these immune cells, which have been artificially modified in vitro. A research team has discovered how to prolong the functionality of CAR-T cells. By inhibiting reductive carboxylation, the team has succeeded in creating CAR-T cells with enhanced immune memory, capable of fighting tumor cells for much longer. These are very promising results, to be read in the journal Nature. Without reductive carboxylation, CAR-T cells do not differentiate as much and retain their anti-tumor function for longer. They even tend to transform into memory T lymphocytes, a type of immune cell that retains a memory of the tumour elements that need to be attacked. The inhibitor used to block reductive carboxylation is a drug already approved for the treatment of certain cancers. It is therefore possible to reposition it in order to extend its use and produce more powerful CART cells in vitro. Of course, its efficacy and safety need to be tested in clinical trials

As industry steps aside, scientists seek innovative ways to make sure expensive treatments can reach people who need them.

Interesting article about the issue of RNA-based vaccines stability. Indeed rings of RNA could be a solution, but there are other strategies also promising and maybe more mature, such as VLPs delivering stable mRNAs or proteins.

By the way, GEG Tech will present in two weeks at the World Vaccine Congress very promising results about a new generation of nanoparticles delivering mRNA for vaccination, comprising and among other data about a very high protection against parasite in a Malaria animal model.