Genetic Engineering in the Press by GEG

The integration of cleavable donor plasmids with hydrodynamic delivery opens up new avenues for treating genetic disorders. Cleavable donor plasmids imply that when the CRISPR-Cas system is directed to a specific location in the genome where a modification is desired, the Cas enzyme not only cleaves genomic DNA at the targeted site, but can also cleave the donor plasmid at the designated sites. This cleavage releases the donor DNA segment, which is then inserted into the genome at the cleavage site via the cell's natural DNA repair mechanisms. In addition, hydrodynamic delivery, a non-viral method, was used for its efficacy and safety compared to viral-based delivery systems. This approach mitigates the risks associated with the host immune response to viral vectors and undesirable mutations due to sustained nuclease expression. The research conducted precise in-frame knock-ins using CRISPR-Cas9 and Cas12a systems, successfully integrating reporter gene cassettes into targeted genomic loci in adult mouse liver. The results demonstrate the system's precise gene-editing capability and its potential applicability in therapeutic contexts. 

The study adapted the novel CRISPR-Cas Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK) based diagnostic platform for species-specific detection of oral bacterial and human pathogens papillomavirus (HPV) nucleic acids. The investigators developed a computer pipeline capable of generating guide RNAs and species-specific genetic primers suitable for SHERLOCK. These constructs were synthesized by cell-free biosynthesis systems, and their specificity and sensitivity were experimentally validated by fluorescence readings of reporter RNAs. The study detected oral bacteria in the single-molecule range that remained specific in the presence of non-targeted DNA found in saliva. In addition, the assay was refined to detect common oral pathogens (e.g. P. gingivalis, F. nucleatum) directly from untreated saliva samples. Detection results, when tested on 30 patient saliva samples, are perfectly aligned with those of other detection methods such as qPCR and 16S rRNA sequencing. This oral pathogen detection method is highly scalable and can be easily optimized for point-of-care implementation. Detection takes around 35 minutes or less, is extremely inexpensive and requires no special skills or techniques.

Recurrent glioblastoma (rGBM) is an aggressive, treatment-resistant brain cancer with no conventional treatment options after chemoradiotherapy. Median overall survival (OS) is less than one year, indicating that effective treatment is an urgent unmet medical need in oncology. Despite limited therapeutic options, chimeric antigen receptor T cells targeting GBM-specific antigens have demonstrated tolerable safety but poor efficacy in adults. In the phase 1 clinical trial of a recent study, the use in cancer patients of intrathecally administered bivalent autologous chimeric antigen receptor (CAR) T lymphocytes targeting the epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2) was associated with early neurotoxicity, possibly the immune effector-associated neurotoxicity syndrome (ICANS). All six patients showed reduction in tumor size and enhancement, but none met criteria for objective response rate. The researchers identified CAR-T cell abundance and cytokine release in all patients, and all developed neurotoxicity. 

Muscular dystrophies are a group of congenital genetic diseases that affect muscle tissue, often resulting in severe disability and considerably reduced life expectancy. Despite intensive research, there is still no effective treatment for patients suffering from muscular dystrophy. It has already been discovered that the muscles that control eye movements are not affected by muscular dystrophy, even in otherwise serious disease processes. In people with muscular dystrophy, the muscles of the body atrophy, while the muscles of the eye remain strong despite the same genetic abnormality. A study has now shed light on this phenomenon. It turns out that a specific gene plays a key role. This gene, fhl2b, is expressed in eye muscles throughout life, but not in other muscles of the body. In the study, researchers used genetically modified zebrafish to investigate how muscular dystrophies affect eye muscles in relation to other muscles in the body. Using Crispr/Cas9 genetic scissors, new models of genetic diseases were created and used on zebrafish.

Belgian researchers from the VIB-KU Leuven Center for Microbiology and the VIB-UGent Center for Plant Systems Biology have extended the toolbox that uses a viral recombinase (Cre) so that it can now specifically recognize, cut and paste multiple DNA sites. The team has identified a set of 16 sites that recombine efficiently with the same site, but not with any other site in that set, for different organisms. This new patented toolbox, complementary to CRISPR, and for some applications going beyond it, is now available to researchers and industrialists in the field of genome engineering. The results are reported today in two simultaneous articles in the prestigious journal Nature Communications.

The FDA has granted priority review to 2 supplemental Biologics License Applications (sBLAs) for lisocabtagene maraleucel (Breyanzi) to expand to new indications to include adult patients with relapsed or refractory follicular lymphoma and relapsed or refractory mantle cell lymphoma (MCL) after exposure to a BTK Inhibitor. Japan's Ministry of Health, Labor and Welfare has also agreed to consider an additional new drug application seeking approval of liso-cel in patients with relapsed or refractory follicular lymphoma. The applications are supported by results from the Phase 2 TRANSCEND FL study (NCT04245839) and the MCL cohort of the Phase 1 TRANSCEND NHL 001 study (NCT02631044). Data from the primary analysis of TRANSCEND FL were shared for malignant lymphoma and showed that liso-cel induced an objective response rate (ORR) of 97% (95% CI, 91.6%-99.4%) and a complete response rate (CR) of 94% (95% CI, 87.5%-97.8%). The results of the primary analysis of the TRANSCEND NHL 001 MCL cohort were also shared. In the primary analysis set (n = 74), liso-cel generated an ORR of 86.5% (95% CI, 76.5%-93.3%) with a CR rate of 74.3% (95% CI, 62.8%-83.8%). 

Genetically modified cavendish bananas that are nearly immune to a devastating fungal disease have been given approval for commercial production and human consumption in Australia.

Human Immunodeficiency Virus (HIV) affects almost 40 million people worldwide, and remains incurable despite considerable efforts to develop new treatments since its discovery in the 1970s. Viral suppression is currently possible thanks to antiretroviral treatments, which can reduce the number of HIV particles in the blood to almost undetectable levels, thus preventing the virus from causing significant damage to patients' immune systems and preventing person-to-person transmission. However, despite the availability of effective antiretroviral treatment, some people living with HIV suffer from HIV-related neurocognitive disorders. These are collectively known as HAND, and have a considerable impact on quality of life. HAND occurs along a continuum of severity; the most severe form is known as HIV-associated dementia (HAD). The exact pathobiological mechanisms underlying HAND are poorly understood, but it is generally accepted that its progression correlates with HIV infection of several cell types in the brain, including astrocytes, microglial cells and macrophages. The emergence of a wide range of gene-editing tools in recent years offers opportunities to develop new, potentially curative therapies against HIV, including therapies aimed at preventing the development of HAND. 

The landscape of cancer research is being transformed by the introduction of new mouse models using prime- and base-editing techniques. The prime-editing mouse model stands out for its high editing efficiencies, particularly in lung and pancreatic organoids. This model facilitates in-depth exploration of drug-resistance mutations, a critical aspect in cancer treatment. Similarly, the base-editing model demonstrates its prowess in creating high-throughput organoid-based models for studying complex genetic variations. The precision and efficiency of these models are crucial to understanding the mechanisms of cancer development and spread, and to identifying potential therapeutic targets.  These advances mark a new era in cancer research, where understanding the genetic underpinnings of cancer is becoming more accessible and actionable.

Probiotics, widely regarded as a treasure trove in the field of microbiology, are currently finding new applications in medicine, animal care and the food industry. However, it is often difficult to use probiotics in their existing form, due to the varying effects of different strains on the health of different individuals. Consequently, it is also difficult to find concrete evidence to support the proposed benefits of probiotics. Fortunately, these challenges can be overcome thanks to the considerable advances we've seen in genetic engineering over the past decade, particularly following the introduction of the hugely popular CRISPR-Cas editing system. By modifying, deleting or introducing specific genes with these tools, we can tailor the activities of probiotic organisms to our health needs. To support the researchers interested in pushing the limits of the field, Professor Nan Peng from the Huazhong Agricultural University, China, along with his colleagues, recently published a review article in Volume 5 of BioDesign Research summarizing the latest advances and hurdles related to the engineering of probiotics with CRISPR-Cas.

For some patients who receive cell therapy such as CAR-T cells, this comes after many other treatments have failed. Researchers noticed that their patients who received the cells while in remission tended to do well after their infusion. They proposed a research study to follow patients who had received a transplant and/or cell therapy. The study included data from 134 patients in the registry who had entered complete remission during the waiting period before receiving their cell therapy. They found that this group of patients had a 43% probability of progression-free survival in the two years following their treatment, roughly the same percentage as patients in the registry who were not in remission when they received CAR-T. Patients in remission had very low levels of toxicities associated with their cell therapies, namely an excessive immune response known as cytokine release syndrome and neurotoxicity, two side effects that can sometimes accompany CAR-T cell therapy. 

The approval of yet another RNA-based vaccine for COVID-19 might not seem momentous. But the endorsement last week by Japanese authorities of a jab against SARS-CoV-2 constructed using a form of RNA that can make copies of itself inside cells — the first ‘self-amplifying’ RNA (saRNA) granted full regulatory approval anywhere in the world — marks a pivotal advance.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer. It is caused by mutations in a large number of genes acquired during a person's lifetime. One of these genes, the tumor suppressor gene TP53, plays a key role. Normally, TP53 helps prevent the development of tumors. However, blood cancer patients with this mutated gene face an extremely poor prognosis, as their genes are resistant to conventional chemotherapeutic agents. Intensive research is therefore being carried out into new therapeutic approaches, such as CAR T lymphocytes, already successfully used for other blood cancers. In particular, an international research team has shown that TP53 mutant AML cells are significantly more resistant to CAR T cell therapy than AML cells without the mutated gene. In their study, the researchers not only examined the mechanism underlying the resistance of mutated AML cells to CAR T cell immunotherapy, but also discovered how the endurance of CAR T cells can be increased, and how a weak point in TP53 mutant AML cells can be exploited to overcome this resistance. 

Researchers have carried out a study of CAR-T cells in breast cancer. However, this therapy doesn't always work on tumors, as their environment suppresses the immune response, and it can also be difficult to find specific features of breast cancer cells to target. To circumvent these challenges, the team directed CAR-T cells at cells surrounding the tumor's blood supply that make the protein endosialin, rather than at actual cancer cells. In experiments with mice, targeting endosialin successfully reduced the growth and spread of breast cancer. The team also tested the treatment on lung cancer tumors in mice and found similar results, suggesting that patients with other types of cancer could benefit from the new treatment too. Moreover, the researchers found that CAR-T therapy does not affect cells without endosialin, indicating that it could work as a cancer-specific treatment with potentially fewer side effects for patients.

Jan Joseph Melenhorst, renowned expert in cancer immunology, emphasizes the importance of understanding the nuances of chronic lymphocytic leukemia (CLL) biology in the development of more effective CAR-T cell therapies. The research reveals significant differences in disease behavior between lymph nodes and blood, which could lead to more targeted treatments. Melenhorst highlights the low sustained response rate to existing CAR-T therapies in CLL, underlining the importance of T-cell health and the need for effective tumor cell activation to improve the efficacy of these treatments. The selection of specific target antigens, such as ROR1, and tumor cell activation are key factors in the design of more effective CAR-T therapies. Melenhorst also discusses the potential of combination therapies and the importance of prior T-cell selection, pointing to promising directions for future research and improved response rates in CLL.

Despite the success of immune checkpoint blockade, a considerable number of melanoma patients do not respond well or suffer relapse after initial success. To find better ways of using CAR T cells to treat melanoma, researchers first looked for an antigen that could be used to target a protein expressed on the surface of cancer cells, but showing lower expression in normal cells. By analyzing three different melanoma datasets, they identified TYRP1, which plays a key role in melanin synthesis, and its surface expression is higher in melanoma cells than in normal tissue. The researchers showed that 60% of acral and mucosal melanoma patients and around 90% of uveal melanoma patients overexpress TYRP1. To investigate the potential new target, the team designed a CAR construct that specifically targeted cells with high TYRP1 expression, and then tested the newly designed CAR T cell in different types of melanoma models. According to their study, CAR T cells completely eradicated cancer cells in cell lines and animal models, showing promising results without inducing toxicity or causing treatment-related adverse effects. The research team plans to launch a clinical trial to evaluate the efficacy of CAR T cells.

Hereditary angioedema (HAE) is a rare genetic disorder characterized by severe inflammatory attacks accompanied by swelling of various organs and tissues. Plasma kallikrein is a protein known to drive multiple inflammatory pathways, including the production of the inflammatory mediator bradykinin, which is overproduced in HAE. Researchers have therefore developed the NTLA-2002 treatment, which is designed to eliminate the target gene kallikrein B1 in hepatocytes. It is administered intravenously as a single dose of Cas9 mRNA and gRNA via lipid nanoparticles. Preclinical studies have demonstrated a sustained and therapeutically relevant reduction in plasma kallikrein activity after a single dose in non-human primates, and the candidate is currently being evaluated in a Phase 1/2 combination clinical trial to assess its safety, tolerability, pharmacokinetics and pharmacodynamics. Clinical data from the Phase 1 dose-escalation portion of this trial showed that a single dose of NTLA-2002 produced robust, dose-dependent and durable reductions in total plasma kallikrein levels, and no serious adverse events were observed. In addition, a reduction in the number of angioedema attacks per month was observed at all dose levels (25 mg, 50 mg or 75 mg).

Researchers at the University of Pennsylvania's Perelman School of Medicine have conducted a study, published in Nature Medicine, which shows that the development of any type of second cancer after CAR T cell therapy is a rare phenomenon. Of 449 patients treated with commercially available CAR T cell therapies at Penn Medicine between January 2018 and November 2023, only 16 were diagnosed with a second cancer after receiving CAR T cell therapy. Most of the secondary cancers (12 of 16) were solid tumors, including skin cancer, prostate cancer and lung cancer. In one patient who developed a secondary lung tumor after CAR T cell therapy, an incidental T-cell lymphoma was also identified in a lymph node removed during surgery for the lung tumor. Molecular analyses showed that the T-cell lymphoma did not harbor the CAR transgene, meaning that it was not a CAR-positive lymphoma and that there was no clear link with CAR T cell therapy.

Anti-CD19 CAR T lymphocytes with a CD28 co-stimulatory domain, such as axicabtagene ciloleucel and brexucabtagene autoleucel, are among the most effective CAR T cell therapies for B-cell non-Hodgkin's lymphoma, but are associated with neurotoxicity in around half of recipients, and cytokine release syndrome (CRS) in up to 90%. Researchers have developed a third-generation autologous anti-CD19 CAR T product, which combines CD28 with a Toll-like receptor 2 (TLR2) co-stimulatory domain. Twenty-one patients with relapsed or refractory B-cell non-Hodgkin's lymphoma were treated in the dose-escalation cohort of a Phase 1 trial and completed the primary follow-up period. No dose-limiting toxicities occurred at the recommended doses. In preclinical studies, the addition of the TLR2 domain maintained or improved efficacy, while reducing production of the pro-inflammatory cytokines IFN-γ and GM-CSF, which are implicated in CRS and associated neurotoxicity syndrome, compared with a CAR with CD28 co-stimulation alone. The absence of neurotoxicity with CD28-based anti-CD19 CAR T cell therapy is remarkable. The addition of the intracellular TLR2 domain with CD28 and CD3ζ alters the cytokine profile of CAR T cells and may explain these clinical results.

The use of BCMA D8 Fab CAR-directed CAR T cell therapy and AUTO8 dual-targeted CAR T cell therapy is safe and feasible in patients with relapsed and refractory multiple myeloma, according to initial results from the ongoing Phase 1 MCARTY trial (NCT04795882). After a median follow-up of 6 months, the objective response rate was 100% in a group of 11 patients who received an infusion. Three patients had a partial response, a very good partial response and a complete response or strict complete response. Overall, progression-free survival was not achieved. In addition, the investigators reported no cases of immune effector cell-associated neurotoxicity (ICAN) syndrome. The investigators aimed to develop a CAR ultra-sensitive to B-cell maturation antigen (BCMA) that co-targets CD19 when using a CAR, as well as to test the product in a clinical study.