Genetic Engineering in the Press by GEG

Despite advances in cancer treatment, colorectal cancer (CRC) remains a major cause of morbidity and mortality worldwide. Although early detection of cancer can improve outcomes, current non-invasive tests, such as fecal immunochemical tests, show limited accuracy. Although effective, invasive diagnostic procedures such as colonoscopy are not suitable for large-scale screening due to their high cost and risk. In addition, colonoscopy screening programs are only available for people over a certain age, usually 45. In a recent study, researchers from the University of California, San Diego, the South Australian Institute of Medical Research and Health and the Colonoscopy Clinic used CRISPR to engineer Acinetobacter baylyi, a non-pathogenic bacterium commonly found in soil, to detect specific DNA mutations released by CRC. The researchers showed that bacteria absorb tumor DNA by natural competence and integrate target sequences into their genome by homologous recombination. This causes a detectable change in the bacteria, enabling sensitive and specific identification of CRC mutations.

The incidence of Parkinson's disease (PD) increases with age and is the second most common neurological disease. PD is caused by the degeneration of dopaminergic neurons in the substantia nigra, which produce dopamine (DA). In particular, astrocytes are essential to the brain's immunological response in Parkinson's disease, as demonstrated by animal models and post-mortem investigations. Tyrosine hydroxylase (Th) is an enzyme present in astrocytes that is crucial for DA production. The optimal approach to managing PD involves the use of levodopa (L-DOPA), a precursor that facilitates DA formation and substitution. This treatment has a limited duration of efficacy, generally extending over five years. In a recent study published in Gene Therapy , researchers use the CRISPR-Cas9 system to induce dopamine (DA) synthesis in the brain of a rat model of Parkinson's disease. The researchers analyzed the rat genome to identify potential guide ribonucleic acid (sgRNA) sequences that were highly specific and did not align with other areas of the rat genome. This led to the identification of 13 sgRNAs for gene activation. Among the 13 th sgRNAs identified in the analysis, the researchers discussed the results of the TH4 sgRNA, as it achieved the highest levels of Th protein expression.

A vaccine developed by the French biotechnology company OSE Immunotherapeutics has demonstrated promising results on advanced lung cancers. Greater effectiveness than chemotherapy with fewer side effects, according to results published this Monday.

Neuro-ophthalmologists have a casual attitude to the perception of their specialty because, for many years, neuro-ophthalmology was strictly a diagnostic specialty. Neurological disorders such as ischemic events, stroke or optic atrophy cause irreversible nerve damage, so neuro-ophthalmologists could help patients by identifying the disease, but not much more. One area of innovation in neuro-ophthalmology is gene therapy and stem cell transplantation. These avenues open up possibilities for treating previously incurable diseases. The development of gene therapy, such as CRISPR-Cas9, has the potential to reverse previously irreversible genetic conditions that affect the optic nerve, mitochondria or other genes. Once a gene's location has been pinpointed, it can be modified using CRISPR-Cas9, and the new gene or gene product can be delivered using a viral vector to transport the new gene into the cell. Gene therapy is not a new concept in ophthalmology. Gene therapy as a treatment for any type of disease was first approved by the FDA for vision. As these therapies progress, they will first take root in the eye, as it is an accessible organ. As more and more therapies enter the neuro-ophthalmologists' toolkit, their role will continue to change.

To develop a phage-based therapy against a diverse range of clinically relevant E. coli, the team tested a library of 162 wild-type lytic phages on a phylogenetically diverse panel of E. coli strains to identify phages with broad and complementary target strain coverage. This state-of-the-art phage screening test led to the identification of eight phages specific to various E. coli strains, displaying complementary binding to bacterial surface receptors and capable of stably transporting inserted cargo. The selected phages were armed with the CRISPR-Cas system containing E. coli-specific sequences. The modified phages were tested in vitro for their ability to inhibit bacterial growth. In addition, the team assessed the in vivo growth inhibitory effect of the phages on E. coli in a mouse model of intestinal colonization by treating the mice with the modified phages and measuring E. coli load. The most effective and complementary CRISPR-Cas-armed phages were selected for further development. The ability of CRISPR-Cas to eliminate E. coli was tested in a biofilm assay. This analysis showed that CRISPR-Cas in modified phages successfully suppressed bacteria in biofilms. Recently, the researchers announced positive interim results from a Phase 1 clinical trial. 

A research team took novel biomaterials to study the effect of tissue mechanics on T cells. A three-dimensional model of the extracellular matrix (ECM) was designed using cells responsible for the different rigidity and viscoelasticity of tissues. The rigidity of an ECM depends on the density of its collagen molecules, while its distinct viscoelasticity depends on the density of collagen molecules cross-linked to each other. This study showed that T cells exposed to a more elastic matrix developed into effector-type T cells, while those exposed to a more viscous matrix became memory T cells. In addition, gene expression analysis revealed the involvement of the AP-1 transcription factor in T lymphocytes' switch from a more elastic, less viscous mechanical environment to a more effector-like gene expression program. When they inhibited one of the components of AP-1 with a drug, the effects of a more elastic collagen matrix on T cells were prevented. Finally, by stimulating CAR-T cells in an elastic matrix, they observed an enhanced ability to kill cancer cells, both in vitro and in vivo. This study opens up new prospects for improving adoptive T-cell therapies by adjusting the mechanics of the biomaterials used. 

The partners in the international CAR T-REX consortium announce the award of a highly competitive EIC Pathfinder Open grant, following the positive evaluation of their project entitled "CAR T Cells Rewired to Prevent EXhaustion in the Tumor Microenvironment". One of 57 projects selected from 858 submissions, with total funding of €2.7m, CAR T-REX was recognized for its radical and ambitious vision to improve the efficacy and safety of CAR T-based cell therapies targeted at solid tumors. Combining innovative methods of genome editing and non-viral gene delivery, CAR T-REX will explore the engineering of transcriptional networks in CAR T cells, to selectively circumvent T cell depletion upon activation in the tumor microenvironment (TME). CAR T-REX aims to construct novel self-regulating genetic circuits controlled by microRNAs in immune cells, use a novel high-performance non-viral gene delivery platform to deliver synthetic miRNA constructs, select and compare the best-performing construct(s) in preclinical settings, and execute GMP-like manufacturing cycles for the final CAR T product. The CAR T-REX consortium brings together a multidisciplinary group of internationally recognized experts and companies from across Europe. 

The US Food and Drug Administration approved the first gene therapy to treat a rare and devastating muscle disease, but limited the approval to kids ages four and five based on currently available evidence.

Currently, no CAR T agents have been approved by the FDA for the treatment of solid malignancies. Several other Phase 1 clinical trials are underway to evaluate the safety and efficacy of CAR T agents, as monotherapy or in combination, in solid malignancies such as glioblastoma, as well as in other cancers. Seeking to replicate the impressive results of CAR-T cell therapies seen in patients with hematological malignancies, researchers launched the BASECAMP-1 trial (NCT04981119) in the hope of identifying patients with advanced solid tumors who would be suitable candidates for treatment in the phase 1/2 EVEREST trial (NCT05736731) evaluating the A2B530 agent. This agent was designed using the novel Tmod logical T cell therapy platform. Tmod agents contain an activating receptor, either a CAR or a T cell receptor, which recognizes an antigen on the surface of tumor cells, specifically CEA in the case of A2B530, and an inhibitory receptor, or blocker, based on the LIR-1 protein, designed to enhance the tumor specificity of the agent. The blocking part of A2B530 exploits the loss of heterozygosity of the HLA-A*02 antigen, one of the most common alleles observed in tumor cells from a US population, to prevent the CAR from affecting healthy tissues. 

Antimicrobial resistance is a major global threat, with nearly five million deaths a year resulting from the failure of antibiotics to treat infection, according to the World Health Organization. Bacteria often develop resistance when resistant genes are transported between hosts. This happens in particular via plasmids, which can spread easily between bacteria and replicate rapidly. This can happen in our bodies and in environments such as waterways. A team of researchers has harnessed the CRISPR-Cas gene-editing system, which can target specific DNA sequences and cut them when encountered. They have designed a plasmid that can specifically target the resistance gene to gentamicin, a commonly used antibiotic. In laboratory experiments, the new research revealed that the plasmid protected its host cell from the development of resistance. In addition, the researchers discovered that the plasmid efficiently targeted antimicrobial-resistant genes in the hosts to which it was transferred, reversing their resistance

Researchers could eventually help develop viable treatments and ultimately a cure for motor neuron disease (MND). They have identified biochemical changes in a protein that is affected by MND. TDP-43 is a protein found in every cell of the body but is particularly important for the health of motor neurons and the brain cells that control voluntary muscle movement. Two research projects have been run, using CRISPR and looking at how TDP-43 proteins become dysfunctional in motor neurons. Researchers found diseased versions of TDP-43 can damage healthy versions of the protein, which may create a cycle of protein dysfunction and degeneration over time. They also discovered that biochemical pathways which control neuron death are triggered early, even before MND symptoms begin. To change the course of the disease, pharmaceutical drugs are needed to be able to prevent neuron death and this TDP-43 protein dysfunction. Researchers are now treating genetically modified mice with MND with different pharmaceutical drugs that specifically target the underlying causes of the disease, and correct the disease mechanism. Their aim is to stop the TDP-43 degenerative cycle and halt the progression of the disease. 

Researchers have engineered stem cells that do not generate dangerous arrhythmias, a complication that has until now thwarted efforts to develop stem cell therapies for injured hearts. Researchers suspected that transplanted stem cells that did not generate arrhythmias were behaving like early embryonic cells chaotically generating signals and causing dangerous heart rhythms. So the researchers used a technique called RNA sequencing to find out which ion channels were being made at different times during cell maturation. Next, the scientists used CRISPR-based genome editing to systematically eliminate depolarizing genes or turn on repolarizing genes, but none of the single-gene edits eliminated the fast heart rhythms. The researchers then undertook a painstaking process of "playing the combinations" by making double and triple gene modifications. Unfortunately, none of these modifications eliminated the arrhythmia. Finally, the scientists created a stem cell line in which three depolarizing genes were eliminated and one repolarizing gene was activated. The generated heart muscle cells were electrically resting, like adult heart muscle, but they contracted when they received an electrical signal to mimic a natural pacemaker. The researchers named these cells "MEDUSA."

The observational BASECAMP-1 study (NCT04981119) has begun screening patients to determine their eligibility for the subsequent phase 1/2 EVEREST-1 study (NCT05736731). BASECAMP-1 (NCT04981119) is recruiting patients with unresectable, locally advanced or metastatic solid tumors expressing carcinoembryonic antigen (CEA) who also show loss of HLA-A*02 heterozygosity, a key feature for the new A2B530 CAR T cell therapy to be effective. EVEREST-1 is the first human study of an agent based on A2B530's novel logic-driven Tmod T cell platform, which enables it to differentiate healthy cells from cancer cells via an activating antigen, CEA, and a blocking antigen, HLA-A*0201. The cell therapy has demonstrated promising preclinical activity thanks to its unique mechanism of action. 

T lymphocytes possess a retinoic acid receptor alpha (RARα) that is known to control gene expression programs in the nucleus, but now also appears to function outside the cell nucleus to coordinate the initial events triggered at the cell surface that lead to T cell activation. The role of RARα has been revealed thanks to the development of CRISPR techniques, advances in imaging and mass spectrometry. The aim of this research is to identify a new pathway, or set of pathways, that could be exploited to control autoimmune diseases and inflammation, or boost immunity to eradicate tumors or fight infections. Researchers had published research showing that retinoic acid (RA) triggers nuclear RARα and the expression of genes important for the differentiation of regulatory T cells. Scientists also knew that RA, present in the blood and taken up by T cells, is then transported to the nucleus by a molecule called cellular retinoic acid-binding protein 2 (CRABP2). CRABP2 in the cytoplasm binds to RA and transports it into the cell nucleus, where it activates nuclear RARα. The researchers set out to understand which other RARα proteins interact with it. This work revealed interactions with the ZAP70 kinase. For T cells, phosphorylation prompts key proteins to act when a threat is near. 

Researchers from one of the largest cancer research and treatment organizations in the USA, have published preclinical research in Nature Communications demonstrating that a CAR T cell therapy targeting TAG72, a target found on the surface of ovarian cancer cells, eradicates cancer cells in mouse models. The researchers also found that by adding the cytokine Interleukin-12 (IL-12), a protein that sends signals to the immune system, to CAR T cell therapy, the treatment was more effective against cancer cells in the laboratory. They also showed that IL-12 also enabled the T cells to both fight the cancer and leave the tumor area, enter the bloodstream and target other cancer cells around the body. The therapy is currently undergoing a first-in-human Phase 1 trial in patients with advanced epithelial ovarian cancer who have already received platinum-based chemotherapy. The trial is testing the safety, side effects and activity of the therapy in patients. The trial is currently recruiting patients for treatment. IL-12 is not currently part of the ongoing Phase 1 clinical trial.

An experimental gene-editing cell therapy for sickle cell disease has shown encouraging initial results in a multicenter Phase 1 and Phase 2 clinical trial. The clinical trial is the first in which CRISPR-Cas12 is used to edit human cells. Cas12 has been shown in preclinical research to be more effective for gene editing as it is a single RNA endonuclease compared with Cas9, which is a double RNA endonuclease. The aim was to make gene editing more precise, more efficient and targeted directly to G1 and G2 hemoglobin, added directly instead of a different activator, so that it mimics exactly what happens in nature with fetal hemoglobin. The four patients in the clinical trial were able to go through all the phases of the study, from mobilization to chemotherapy and perfusion of the CD34-modified cells. They were then transplanted, and the patients can now be transfusion-independent. So far, all four patients have experienced no pain attacks, and any side effects noted were mainly related to the busulfan used in the chemotherapy regimen. All four patients were able to achieve a fetal hemoglobin of over 40% in around 3-4 months. They all had hemoglobin in the normal range from about 3 months post-transplant. Hemolysis markers also normalized, which is a major finding.

A new study highlights the limits of gene editing in human embryos. Researchers have used CRISPR-Cas9 in an attempt to correct genetic errors in human embryos. However, embryonic cells have difficulty repairing broken DNA strands, leading to further mutations. In one study, researchers fertilized donated eggs with donated sperm using intracytoplasmic sperm injection (ICSI) to create 84 embryos. In 33 of the embryos, they used CRISPR-Cas9 to create breaks in the two strands that make up the DNA molecule. However, only nine percent of the targeted sites were repaired using the clinically useful process of homology-directed repair. Fifty-one percent of the broken DNA strands underwent non-homologous end-joining, producing mutations where the strands were reconnected. The remaining 40% of broken DNA strands were not repaired, leading to the loss or duplication of large chunks of chromosome from the site of the break to the chromosome end. Abnormalities of this kind affect the viability of embryos. The researchers point out that this raises concerns about the use of gene editing in human embryos, particularly to eliminate serious hereditary diseases. Technical improvements are needed to reduce the risks and increase the efficiency of gene editing in embryos.

Antimicrobial resistance (AMR) is a generic term that describes when microbes, mainly bacteria or fungi, become resistant to the drugs used to treat them. AMR is a complex global problem because it is bacteria that become resistant to antibiotics, and bacteria can exchange genetic material that makes them resistant, giving rise to "superbugs" that cannot be treated with any type of antibiotic. This means that the misuse and overuse of antibiotics in one part of the environment or one part of the world can have global impacts. By 2050, it is estimated that antimicrobial resistance will cause more human deaths than cancer and diabetes combined. Researchers have studied CRISPR-Cas9 with the aim of using it to eliminate antibiotic resistance genes. Cas9 could be programmed to target an antibiotic resistance gene, meaning that bacteria that received this CRISPR-Cas9 treatment would lose the targeted antibiotic resistance gene and could be treated with antibiotics again. The CRISPR-Cas9 delivery plasmid they chose, pKJK5, is known to have a very broad host range, meaning it can be taken up by many different species of bacteria.There are still several technical challenges they face, one of which is delivery efficiency.

The aim of immunotherapy strategies is to use cells from the patient's own immune system to destroy tumor cells. CAR T cell therapy is an effective immunotherapy for the treatment of blood cancer. Around 35,000 people are affected by blood cancer in France every year, with 1.24 million cases worldwide. By closely studying some of the immune cells generated during this therapy, known as CD4 T cells, scientists have discovered that these cells are able to neutralize tumor cells at a distance by producing interferon gamma (IFN-γ). To reveal the novel mechanism of action of these remote killer cells, scientists first explored preclinical models to analyze the mechanism in detail, notably using in vivo imaging techniques, and then verified the relevance of the results on patient samples. This study raises new hopes for blood cancer patients with an incomplete response to CAR T cell therapy, and for IFN-γ-sensitive cancers.

The Agriculture Department granted approval to cultivated meat producers for the first time in the United States, representing a watershed moment for the alternative protein industry.

A type II variation application seeking approval of the ciltacabtagen autoleucel (Carvykti, cilta-cel) in adult patients with relapsed multiple myeloma refractory to lenalidomide has been submitted to the European Medicines Agency. The application is based on the results of the phase 3 CARTITUDE-4 trial (NCT04181827), with data showing that CAR T-cell therapy reduces the risk of disease progression or death compared with pomalidomide (Pomalyst), bortezomib (Velcade), and dexamethasone (PVd) or daratumumab (Darzalex), pomalidomide and dexamethasone (DPd) in people with relapsed or refractory disease who have received 1 to 3 prior lines of therapy. Median progression-free survival (PFS) has not yet been achieved with cilta-cel versus 12 months with the control regimen. In addition, PFS rates at 12 months were 76% in the experimental group and 49% in the control group. CAR T-cell therapy elicited an overall response rate of 88% versus 67% with control. Notably, 73% of patients who received cilta-cel had a complete response to treatment versus 22% who received control. Data will be shared in a special session at the next ASCO annual meeting.

Stargardt's disease is an inherited macular neurodegenerative disorder that causes retinal dystrophy and vision loss. Stargardt disease is caused by mutations in the ABCA4 gene, which encodes a protein involved in the visual cycle and the transport of toxic photoproducts out of the retina. Currently, there is no treatment for Stargardt's disease, and although restoring ABCA4 expression may help prevent or treat the retinal dystrophy of Stargardt's disease, gene replacement therapy has not yet succeeded in preventing retinal dystrophy or improving vision. In a recent study, a team of researchers used the CRISPR-Cas9 system to modify pathogenic ABCA4 mutations in human-induced pluripotent stem cells (iPSCs) from two patients with Stargardt disease. This approach led to successful correction of both pathogenic ABCA4 variants without causing detectable off-target effects. Siles also noted that the use of single-stranded oligodeoxynucleotides as donor templates for CRISPR-mediated ABCA4 editing ensured permanent restoration of the ABCA4 sequence. However, as this approach is still in its infancy, a further increase in the specificity and precision of gene editing is crucial. The team also plans to validate the feasibility of this method using in vitro 3D retinal cultures. 

Researchers have designed a new nanoparticle sensor that could enable early diagnosis of cancer with a simple urine test. The sensors, which can detect many different cancerous proteins, could also be used to distinguish the type of a tumor or how it is responding to treatment. The nanoparticles are designed so that when they encounter a tumor, they shed short sequences of DNA that are excreted in the urine. Once the sensors are secreted in the urine, the sample can be analyzed using a paper strip that recognizes a reporter that is activated by a CRISPR enzyme called Cas12a. When a particular DNA barcode is present in the sample, Cas12a amplifies the signal so that it can be seen as a dark strip on a paper test. In tests in mice, the researchers showed that they could use the sensors to detect the activity of five different enzymes that are expressed in tumors. They also showed that their approach could be scaled up to distinguish at least 46 different DNA barcodes in a single sample, using a microfluidic device to analyze the samples. The researchers designed their test so that it can be performed using a strip of paper which they hope could make it affordable and accessible to as many patients as possible.