Researchers have developed a reverse genetics system for the African swine fever virus (ASFV). The scientists construct synthetic DNA, a laboratory-made version of the virus’s genetic material. Fragments of ASFV are then modified and assembled into complete genomes in yeast using its natural recombination mechanism. The modified genomes are subsequently transferred to E. coli, allowing them to be isolated in larger quantities. The synthetic DNA is then transfected (artificially introduced) into mammalian host cells, which are subsequently infected by a helper virus. This helper virus is an attenuated version of ASFV, modified using CRISPR/Cas9 technology—a powerful genetic editing tool capable of cutting DNA at precise locations. These alterations prevent the helper virus from replicating on its own. Despite this inhibition, the helper virus still provides the proteins and machinery necessary for the replication and assembly of the synthetic DNA into new viral particles. This process enables the production of live recombinant viruses containing the specific genetic modifications introduced into the synthetic DNA. The new system will help researchers develop vaccines and study the pathogenesis and biology of ASFV, a deadly and highly contagious viral disease affecting domestic and wild pigs.

Chimeric Antigen Receptor T-cell (CAR-T) therapy has revolutionized cancer treatment by engineering a patient's T-cells to target and eliminate malignant cells. However, certain cancers still evade detection, leading to treatment resistance and relapse. American researchers have recently introduced an innovative approach known as ALA-CART (adjunctive LAT-activating CAR-T cells), designed to overcome these challenges. In preclinical studies, ALA-CART demonstrated enhanced efficacy against acute lymphocytic leukemias that were unresponsive to traditional CAR-T therapies. By optimizing T-cell activation and targeting capabilities, this next-generation therapy not only improved cancer cell eradication but also showed potential in reducing associated side effects. These findings suggest that ALA-CART could offer a more robust and durable treatment option for patients with resistant cancers, marking a significant advancement in the field of immunotherapy.

This year’s Breakthrough Prize in Life Sciences has been awarded to David Liu, a biochemist at Harvard University, for the development of base editing and prime editing, two novel genome editing technologies. The Breakthrough Prize in Life Sciences honors researchers whose work has improved scientific understanding of living systems and contributed towards extending human life.

Primary hyperoxaluria (PH) is a group of rare, autosomal recessive metabolic diseases characterized by excessive oxalate production by the liver and subsequent accumulation of oxalate in the kidneys and other organs. Type I PH (PH1), the most common and severe subtype, is thought to affect 1 to 3 people per 1,000,000 in the general population. PH is incurable, and current therapeutic approaches focus on relieving symptoms and preventing the accumulation of oxalate in the kidneys and blood vessels. However, Arbor Biotechnologies has developed a treatment for PH1, called ABO-101, and announced in a recent press release that the FDA has granted both Orphan Drug Designation and Rare Pediatric Disease Designation to this treatment. ABO-101 is a novel investigational gene-editing therapy designed as a single liver-directed treatment that permanently deactivates the HAO1 gene to reduce PH1-associated oxalate production. The candidate uses a lipid nanoparticle delivery system (licensed from Acuitas Therapeutics) encapsulating messenger RNA expressing a CRISPR Cas12i2 type V nuclease and an optimized guide RNA targeting the HAO1 gene. 

Chronic diseases such as cancer and chronic infections often leave the immune system in a state of exhaustion, where its front-line defenders, the T lymphocytes, lose their ability to function effectively. Research has identified a rare type of immune cell, called a T stem cell, which holds the key to maintaining robust, long-term immune responses. The study reveals that the stamina of these stem-type T lymphocytes is fuelled by a protein called ID3, expressed by a gene of the same name. These ID3+ T cells have a unique ability to self-renew and resist depletion, allowing them to maintain immune responses for much longer than other T cells that do not express ID3. The study also revealed that specific signals in the body could increase the number of ID3+ T cells, paving the way for improved treatments such as CAR T cell therapy. Although CAR T therapy has been transformative in treating certain cancers, its effectiveness can diminish over time due to T cell depletion.

Researchers at the University of Tokyo have developed a groundbreaking CRISPR-based barcoding system to study small extracellular vesicles (sEVs), nanosized particles crucial in intercellular communication and disease progression. The system uses CRISPR gene-editing technology to introduce RNA barcodes into sEVs, allowing thousands of genes to be analyzed simultaneously in a single pooled experiment. CIBER (CRISPR-assisted individually barcoded extracellular vesicle-based release regulator) offers detailed insights into sEV subpopulations, enabling more efficient and comprehensive studies than conventional methods. This innovation opens new pathways for sEV-based diagnostics and therapeutic applications. 

In the quest to improve the efficacy of CAR-T cell therapy against hepatocellular carcinoma (HCC), a recent study has identified a pivotal role for CD39 expression in modulating CAR-T cell function. The research, conducted by a team of scientists, investigated the impact of CD39 modulation on CAR-T cells, hypothesising that optimal levels of CD39 could increase the therapeutic potential of these cells against HCC. The study began with the isolation and culture of primary human T lymphocytes, transduced with a lentiviral vector encoding the CAR CD39 construct. The study also used a combination of in vitro and in vivo experiments, including a mouse model of subcutaneous HCC, to demonstrate that CAR-T cells with moderate levels of CD39 expression displayed superior antitumour activity to those with high or low levels of CD39. In addition, the study explored the use of mdivi-1, a small molecule inhibitor, to modulate CD39 expression, revealing a synergistic effect when combined with CD39 suppression that significantly improved the antitumour response. 

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. 

Recent developments in allogeneic CAR-T cell therapy have shown promise in treating relapsed or refractory large B-cell lymphoma (LBCL). Californian biotech Allogene Therapeutics reported encouraging results from their Phase 1 ALPHA and ALPHA2 trials, evaluating the efficacy of ALLO-501, an allogeneic CAR-T product, in LBCL patients. The trials demonstrated complete response rates of 67% and 58%, respectively, with a median duration of response of 23.1 months among responders. Notably, patients with minimal disease burden at treatment initiation exhibited particularly favorable outcomes. These findings suggest that allogeneic CAR-T therapies could serve as effective "off-the-shelf" treatments, offering a more accessible and timely alternative to autologous CAR-T therapies, which require individualized cell harvesting and processing. Pitfalls however include accepting the “non-self” and graft versus-host disease (GvHD). An ongoing third trial aims to further assess the potential of this approach to redefine standard care practices in oncology

Recent developments in CRISPR-Cas9 gene-editing technology have significantly advanced the field of regenerative medicine. CRISPR-Cas 9 offers precise and efficient methods for tissue repair and the treatment of genetic disorders. It enables targeted genome modifications, including gene knock-ins, knockouts, and base conversions, facilitating the correction of mutations responsible for diseases such as cystic fibrosis, sickle cell disease, and osteogenesis imperfecta. Beyond genetic corrections, CRISPR is instrumental in reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which can then be differentiated into specific cell types for therapeutic applications. It is also a key research tool facilitating genetic screening and disease models creation. While challenges remain, especially with safe delivery and minimizing off-target effects, the potential is undeniably vast. These advancements underscore CRISPR's pivotal role in enhancing tissue repair processes and developing innovative treatments for previously intractable conditions. 

CAR T cell therapy is a personalized form of immunotherapy that uses a deactivated virus to program an individual's T cells to target and kill their cancer cells. Since the first CAR T cell therapy was approved in 2017, more than 30,000 patients with blood cancers have been treated. Some of the first patients treated in clinical trials have experienced durable remissions of a decade or more. In late 2023, the FDA announced that it was investigating several reported cases of secondary T-cell malignancies in patients who had previously received CAR T cell therapy products. In 2024, the FDA also began requiring drug manufacturers to add a safety warning to the label of CAR T cell products. As a result, researchers analysed samples from 783 adult and paediatric patients in Philadelphia who had been treated with CAR T cell therapy in clinical trials and found 18 cases of secondary cancers. None of the 18 cases showed evidence that they were caused by insertional mutagenesis. The researchers attributed the rarity of secondary cancers to immune system suppression due to previous anti-cancer treatments.

Although clinicians have successfully used CAR-T cells in cancer, many patients experience a cancer relapse caused by the hostile environment created by the cancer cells. After repeated encounters with cancer cells, CAR-T cells lose their ability to effectively divide and attack the tumour. Therefore, a research team used CRISPR screening to identify potential genes that could improve CAR-T cell therapy. The researchers found that knocking out the CUL5 gene improved the growth and longevity of CAR-T cells, even after repeated exposure to cancer cells. The gene of interest, CUL5, is involved in the degradation of specific proteins inside cells. When this gene is inactive, a cell signalling pathway known as the JAK-STAT signalling pathway becomes more sustained. This pathway sends signals encouraging T lymphocytes to grow and multiply, making them more effective at fighting cancer. Therefore, a new way of partially reducing CUL5 activity has been developed by using a virus to deliver genetic material to CAR-T cells.

CAR-T therapy treatments have proven effective in patients with blood tumors, however, their efficacy in solid tumors has been limited due to a variety of factors, including the tumor microenvironment. For this reason, researchers used a 3D microfluidic model made from patient-derived organotypic tumor spheroids (PDOTS) to study the mechanisms of treatment resistance of CAR-T cells designed in particular to target B7-H3, a common antigen in solid tumor cancers. By inhibiting the function of TBK1, a gene previously associated with immune evasion, they were able to restore CAR-T cell activity, prevent dysfunction and increase T cell proliferation. The researchers also found that inhibiting or suppressing TBK1 made cancer cells more sensitive to immune cell targeting and killing. The results therefore suggest that targeting TBK1 could reduce treatment resistance and improve CAR T efficacy in solid tumor cells expressing B7-H3, and also demonstrate the feasibility and utility of using PDOTS to study tumor-immune cell interactions. 

Nature Medicine asks leading researchers to name their top clinical trial for 2025, from gene therapies for prion disease and sickle-cell disease to digital tools for cancer and mental health.

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.