Genetic Engineering in the Press by GEG

Neuroblastoma (NB) is a fatal childhood cancer that does not respond well to checkpoint immunotherapy due to low immunogenicity caused by low MHC class I expression and low neoantigen load. Researchers found that CRISPR-mediated silencing of the ERAP 1 (Endoplasmic Reticulum AminoPeptidase 1) gene in combination with entinostat, a histone deacetylase inhibitor, increased the immunogenicity of NB cells, making them more sensitive to PD-1 treatment. ERAP is an enzyme that cleaves peptides before loading them onto MHC class I molecules, and its inhibition leads to the generation of new antigens capable of inducing robust anti-tumour immune responses. The team observed that although none of the strategies was compelling, a triple combination of ERAP 1 silencing + Entinostat + PD-1 blockade significantly improved toxicity-free survival rates in a mouse model of NB. The approach is promising because it could help overcome the low immunogenicity of NB, making it more sensitive to immunotherapy.

The CRISPR tool is very powerful, but it has side effects. By modifying one gene, CRISPR can activate or deactivate many associated genes, leading to unexpected results. To combat these side effects, a team of researchers uses computer modeling and deep learning to predict the broader impacts of CRISPR gene modifications on the genome. The model allows the team to simulate the effects of modifying a single gene on the whole genome, enabling them to predict and avoid unforeseen consequences. It also contributes to the evaluation and identification of new genetic targets. Their approach has far-reaching implications in various fields, including cancer treatment and musculoskeletal applications. For example, the team has identified candidate genes that can enhance the differentiation of induced pluripotent stem cells into more effective cancer-fighting cells. Furthermore, CRISPR must penetrate the cell's nucleus using nanoparticles to function. However, the researchers realized that the nanoparticles can negatively affect the cells. They, therefore, used various computer modeling techniques to predict how these mechanisms affect the ability of stem cells to differentiate and survive.

Scientists use a long, thin glass needle to inject eggs or embryos, a technique known as microinjection, which requires a great deal of time and expertise. However, a new delivery approach has been inspired by an egg yolk protein found in most animals, called vitellogenin, which provides an energy source for the growing egg. Vitellogenin is a large protein, but the team isolated the small part that binds to the receptor on the surface of the egg. This is a very small tag (around 10 amino acids) to which various fillers, such as Cas9, can be added. The team has successfully used VitelloTag on two distantly related species: the starfish (Patiria miniata) and the acorn worm (Saccoglossus kowalevskii). However, microinjection remains the method of choice for delivering CRISPR-Cas9 to many organisms. Penetration (the percentage of cells that successfully absorb the CRISPR load) can reach 90% with microinjection, while with VitelloTag, the team achieved around 30% penetration in starfish and acorn worms. 

CAR-T cells are highly effective in the treatment of certain blood cancers. However, this new therapy, first approved in 2017 in the United States and a year later in Europe for treating acute lymphoblastic leukemia (ALL), still poses challenges. For example, there are no effective CAR-T cell-based therapies for solid tumors. In addition, remissions induced by CAR-T cells are not always long-lasting, and the production of CAR-T cells is slow and laborious. Researchers wanted to find a solution to these limitations using an advanced CRISPR method to increase the efficacy of cancer immunotherapies. The project is called Prime-CAR Inspection: ‘Prime’ refers to the method that enables the targeted and programmable incorporation of DNA modifications into therapeutic T cells; ‘CAR’ refers to the chimeric antigen receptor; ‘Inspection’ refers to the validation of the safety of new gene-editing methods using advanced molecular diagnostics. The new CRISPR Prime Editing method in the study requires only a single strand break, enabling more precise genome modifications. The German Research Foundation (DFG) supports this research project with almost two million euros over the next six years as part of the Emmy Noether program.

Currently, three CAR-T cell therapies (axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel) and lisocabtagene maraleucel (liso-cel)) are approved for the treatment of Diffuse Large B-Cell Non-hodgkin's Lymphoma (DLBCL). However, patients receiving these therapies are at high risk of developing either Cytokine Release Syndrome (CRS) or Immune effector Cell-Associated Neurotoxicity Syndrome (ICANS). To monitor and manage these toxicities, the US Food and Drug Administration (FDA) has implemented a Risk Evaluation and Mitigation Strategy (REMS) requiring CAR-T therapy recipients to remain within two hours of their Authorized Treatment Center (ATC) for four weeks after therapy and to refrain from driving for eight weeks after treatment. However, a recent study showed that most patients who developed CRS or ICANS did so within the first two weeks after infusion. This would justify a shorter and more flexible toxicity monitoring period. The results also showed that after two weeks, infections, which developed in 14.5% of patients within 28 days of infusion, were the most common cause of death. This underlines the fact that infection is the main challenge following CAR-T treatment.  

Researchers have refined the primary editing (PE) method for correcting the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a significant cause of cystic fibrosis. They combined several efficiency improvements, including engineered PE guide RNAs and PEmax architecture. They achieved high correction efficiency in immortalized bronchial epithelial cells and substantial correction in patient-derived airway epithelial cells. In addition, they used strategic silent modifications to evade cellular mismatch repair and incorporated PE6 variants and dead simple guide RNAs (dsgRNAs) to improve targeting and editing efficiency. Results showed that these combined optimizations significantly increased the correction efficiency for CFTR F508del, reaching 58% in immortalized bronchial epithelial cells and 25% in primary airway epithelial cells derived from cystic fibrosis patients. Functional analyses indicated that corrected CFTR ion channels restored more than 50% of wild-type function, comparable to current drug treatments for cystic fibrosis. 

Nineteen genetic therapies have been approved by the U.S. Food and Drug Administration (FDA) to date, a number that now includes the first CRISPR genome editing therapy for sickle cell disease, CASGEVY (exagamglogene autotemcel). This extraordinary milestone is widely celebrated because of the promise for future genome editing treatments of previously intractable genetic disorders and cancers. At the same time, such genetic therapies are the most expensive drugs on the market, with list prices exceeding $4 million per patient.

CAR-T therapy, which harnesses a person’s own immune cells, racks up some astonishing success stories against deadly brain tumours in children. 

Activated T cells in patients with autoimmune diseases, cancer or viral infections expose large amounts of B7H6 on their surface. Co-culture experiments in the culture dish have shown that Natural Killer (NK) cells recognize activated T cells by their B7H6 expression. In contrast, T cells whose B7H6 gene was knocked out with CRISPR-Cas were protected from lethal NK cell attack. B7H6 can now be classified as another inhibitory immune checkpoint on T cells. Researchers have thus discovered that NK cells can thus alter the effect of cancer therapies using immune checkpoint inhibitors (ICIs). They may also be responsible for the rapid decline of therapeutic CAR-T cells. Interventions in this mechanism could potentially improve the efficacy of these cellular cancer immunotherapies. 

The European Commission has approved ciltacabtagene autoleucel (cilta-cel; Carvykti) for the treatment of patients with relapsed or refractory multiple myeloma who have received one or more previous lines of therapy (such as an immunomodulatory drug and a proteasome inhibitor), have progressed on their last line of therapy and are refractory to lenalidomide Revlimid. The expanded indication for cilta-cel was based on the results of the phase 3 CARTITUDE-4 trial (NCT04181827), which demonstrated that after a median follow-up of 15.9 months (range: 0.1-27.3), treatment with cilta-cel reduced the risk of progression or death by 74% compared with standard therapies in patients with relapsed or refractory disease who had received 1 to 3 prior lines of treatment (HR: 0.26; 95% CI, 0.18-0.38). Median progression-free survival (PFS) was not yet achieved in the cilta-cel arm versus 11.8 months (95% CI, 9.7-13.8) in the standard of care (SOC) arm. Estimated 12-month PFS rates were 76% (95% CI, 69%-81%) with cilta-cel versus 49% (95% CI, 42%-55%) with SOC.

The rise of RNA viruses such as SARS-CoV-2 highlights the need to find new ways of combating them. RNA targeting tools such as CRISPR/Cas13 are powerful but ineffective in the cytoplasm of cells, where many RNA viruses replicate. Scientists have developed a solution: Cas13d-NCS, a new system capable of transferring nuclear CRISPR RNAs (crRNAs) into the cytosol. crRNAs are short RNA molecules that guide the CRISPR-Cas complex to specific target sequences for precise modifications. In the cytosol, the protein/cRNA complex targets complementary RNAs and degrades them with unprecedented precision. With remarkable efficiency, Cas13d-NCS outperforms its predecessors in degrading mRNA targets and neutralizing self-replicating RNA, including the replication sequences of Venezuelan Equine Encephalitis (VEE) RNA virus and several variants of SARS-CoV-2, unleashing the full potential of Cas13d as a programmable antiviral tool. This new molecular tool enables crRNA molecules located in the nucleus of a cell to move into the cytoplasm, making it highly effective in neutralizing RNA viruses. This breakthrough opens the door to precision medicine and proactive viral defense strategies. 

In a study, researchers have developed a novel CRISPR-Cas9 delivery system using cryo-choked lung tumor cells to target Non-Small Cell Lung Cancer (NSCLC), particularly those with KRAS mutations. The technique involves using lung tumor cells treated by rapid shock with liquid nitrogen, effectively inactivating their pathogenicity while preserving their structure and surface receptors. These cryo-choked cells serve as carriers for CRISPR-Cas9, which aims to edit the CDK4 gene, a key player in the proliferation of KRAS-mutant NSCLC cells. In NSCLC carrier mice, in vivo injection of packaged CRISPR-Cas9 plasmids into cryo-choked lung cancer cells led to an almost four-fold higher concentration in the lungs compared with administration by lipofectamine. The new CRISPR delivery system showed a significant reduction in tumor size and prolonged survival, marking an important advance in the use of gene editing for cancer treatment.

Virologist Beata Halassy says self-treatment worked and was a positive experience — but researchers warn that it is not something others should try. 

Duchenne muscular dystrophy (DMD) is a rare, incurable muscular disease affecting around 1 in every 3,500 to 5,000 male births worldwide. The disease follows an X-linked pattern of inheritance, and exonic duplications of the gene coding for DMD dystrophin are frequently observed in DMD patients. Dystrophin is a cytoplasmic protein that plays a mechanical role in muscle. In a recent study, French researchers used the CRISPR-Cas9 gene-editing technique to target intronic regions of the DMD gene. Their aim was to delete certain duplicated regions in patients' immortalized myogenic (muscle progenitor) cells, in particular duplications of exon 2, exons 2 to 9 or exons 8 to 9, which are known hotspots for mutations in DMD patients.They confirmed restoration of the DMD open reading frame and rescued dystrophin expression by Western blotting and mytotube immunostaining after CRISPR-based deletion of the target duplications. RNA sequencing suggested gene rescue in dystrophin-related pathways. Off-target analysis based on predicted nearby off-targets revealed no significant unintended genetic changes at these loci.

Synovial sarcoma is diagnosed in fewer than 1,000 people in the United States each year. This cancer can develop in the extremities or in the soft tissues of the abdomen or lungs. It occurs most often in young adults. It is slightly more common in men than in women. New treatments for this cancer are currently being developed. Moreover, accelerated approval for a treatment for adults with synovial sarcoma, afamitresgene autoleuecel immunotherapy (Tecelra ®, also known as afami-cel), has been granted by the US Food and Drug Administration (FDA). The cancer-causing protein targeted by afami-cel is called MAGE-A4. It is the first modified T-cell therapy to receive FDA approval for a solid tumor cancer. The clinical trial was an international study involving 52 people with synovial sarcoma and Myxoid/Round Cell LipoSarcoma (MRCLS), another type of soft tissue sarcoma. These patients had not responded to other treatments. Around 37% of patients saw their tumors shrink after receiving a single dose. The drug helped around 39% of people with synovial sarcoma and 25% of those with MRCLS.

Stem-cell transplants have freed seven people of the virus, but researchers say most long-term interventions remain a distant prospect.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare disease with sudden onset symptoms, including nerve damage affecting movement, sensation, speech, breathing, and heart rhythm. Treatments such as glucocorticoids, plasmapheresis, and intravenous gamma globulin (IVIG) can help manage symptoms but cannot completely eradicate the disease. However, a research team has reported using BCMA-CD19 bispecific CAR-T cells to treat relapsed/refractory CIDP, restoring the balance of immune responses by temporarily and profoundly eradicating B cells and plasma cells. After being treated with this therapy, one patient made significant progress in functioning according to the INCAT disability and MRC scores. Remarkably, almost complete recovery of muscle power was observed 180 days after administration of CAR-T, along with the ability to walk again. This study, therefore, highlights the evolution of patients' symptoms after treatment and confirms the safety of CAR-T cell therapy for CIDP. 

In a recent study of 30,000 patients treated with CAR-T therapy, second cancers reported are rare. However, the US FDA has monitored around 25 cases of CAR-T cell second cancers, generally lymphomas. Furthermore, researchers do not know how the CAR-T cells became a lymphoma. It is possible that the cells had lymphoma-causing mutations when they were initially collected from the patient and that CAR-T treatment caused the activation and expansion of these cells, that the mutations may have occurred when the CAR-T cells were prepared outside the patient, or that the CAR-T cells may have acquired the mutations after being returned to the patient. For future diagnostic and treatment purposes, the researchers note that switchable cell therapies could allow patients to take a drug to adjust the CAR-T cell activity daily, thereby reducing toxic side effects. 

According to a 2020 report by the Centers for Disease Control and Prevention (CDC), Florida has the second highest number of new cases of melanoma per year in the United States. However, these patients will soon have access to the first cellular therapy for this advanced form of skin cancer, following its recent approval by the Food and Drug Administration (FDA). This therapy, known as tumor-infiltrating lymphocyte therapy, or TIL, uses patients' own immune cells to fight their cancer. Trials testing TIL therapy, called lifileucel, prior to FDA approval showed a 32% response rate among 153 patients, and more than half of patients who responded favorably to treatment had maintained that response for more than three years. Lifileucel is approved for patients whose melanoma has progressed despite treatment with other forms of immunotherapy, including those involving checkpoint inhibitors, and targeted therapy for melanomas with a common BRAF gene mutation. 

Two blood biomarkers, pre-lymphodepletion C-reactive protein and ferritin, can help predict which lymphoma patients are at risk of poor outcomes after CAR-T cell therapy. Researchers analyzed data from 136 patients with relapsed or refractory diffuse large B-cell lymphoma. Almost all (93%) developed cytokine release syndrome, and 61% developed immune effector cell-associated neurotoxicity syndrome (ICANS). The researchers determined that patients with baseline blood levels of C-reactive protein (CRP) of at least 4 mg/dL and baseline ferritin of 400 ng/mL or more faced a higher risk of adverse outcomes, including shorter PFS, shorter OS and higher rates of severe toxicities. However, given that patients in the “low-risk” category are much less likely to experience severe toxicities or any type of major morbidity or mortality, these patients could be treated with CAR-T on an outpatient basis.

In the never-ending quest to discover previously unknown CRISPR gene-editing systems, researchers have scoured microbes in everything from hot springs and peat bogs to poo and even yogurt. Now, thanks to advances in generative artificial intelligence (AI), they might be able to design these systems with the push of a button.

This week, researchers published details of how they used a generative AI tool called a protein language model — a neural network trained on millions of protein sequences — to design CRISPR gene-editing proteins, and were then able to show that some of these systems work as expected in the laboratory1.