Genetic Engineering Publications - GEG Tech top picks

Glioblastoma (GBM) is the most common and aggressive type of cancerous brain tumor in adults. People with GBM generally expect to live 12 to 18 months after diagnosis. Despite decades of research, there is no known cure for GBM, and treatments have only a limited effect on extending an individual's life expectancy. However, researchers have tested a technology that delivers CAR-T cells targeting two proteins commonly found in brain tumors: epidermal growth factor receptor (EGFR), estimated to be present in 60% of all GBMs, and interleukin-13 receptor alpha 2 (IL13Rα2), which is expressed in over 75% of GBMs. While CAR-T cell therapy for blood cancers is usually administered intravenously, the researchers administered these dual-targeted CAR-T cells intrathecally, by injection into the cerebrospinal fluid, so that the modified cells could reach the tumors more directly in the brain. Magnetic Resonance Imaging scans taken 24 to 48 hours after administration of dual-targeted CAR-T cells targeting EGFR and IL13Rα2 revealed a reduction in tumor size in all six patients, and these reductions were maintained up to several months later in a subgroup of patients. 

In a review, researchers detail the development history of Principal Editing (PE), the latest evolution of CRISPR-Cas-based technologies. PE was proposed by a team of researchers in 2019, which is characterized by the absence of double-strand breaks (DSBs) or homology sequence patterns with variable application scenarios, including point mutations as well as insertions or deletions. The PE system consists of two parts: the master editors (PEs) and the master editing guide RNA (pegRNA). This PE system has developed and progressed rapidly over the last four years, with versatile advances in its architecture to increase editing efficiency, targeting and specificity, including a new pegRNA design, PE modification and improved delivery. Moreover, despite its relatively recent inception, PE has been widely applied to correct pathological mutations associated with genetic diseases, both in vitro and in vivo , presenting great potential for advancing the field of gene therapy from bench to bedside.

Researchers set out to develop a robust off-switch for the highly efficient Cas3 system they had previously discovered from Neisseria lactima, a bacterium that lives harmlessly in the human upper respiratory tract. Examining all known anti-CRISPRs that have been reported in the literature as inhibitors of other Cas3 variants from distinct bacterial organisms, they found two, AcrIC8 and AcrIC9, with a strong cross-reactive effect against Neisseria Cas3. Using genetic and biochemical studies at UM and cryogenic electron microscopy analyses at Cornell, they determined the mechanism of action and structure of AcrlC8 and AcrlC9 at the molecular level. Both proteins prevent the CRISPR-Cas3 machine from binding to its DNA target site, but by slightly different mechanisms. Finally, the team provided key proof-of-concept that each of these anti-CRISPR proteins can act as a switch for CRISPR-Cas3 in human cells. They can almost completely block two versions of CRISPR-Cas3 technologies, one for deletion of important genomes and the other for gene activation, making them the first switches developed for any CRISPR-Cas3 gene editor. 

Traditional CAR-T cells, while effective against liquid cancers, face challenges in solid tumors: the cells wear out and ultimately fail to destroy the cancer completely. Ground-breaking research is providing an innovative approach to this challenge. Researchers are introducing CAR-T cells that excrete the IL-10 molecule. In other words, the cell has been designed to produce its own "drug" to stay healthy in the tumor's hostile environment. In the laboratory, this innovative CAR-T therapy systematically eradicated cancerous tumors in mouse models. What's more, in ongoing clinical trials, eleven patients have appeared to achieve complete remission with this treatment, representing a 100% success rate to date. Notably, the evidence from the laboratory study suggests the long-term efficacy of the therapy, and indicates that its manufacture could be both faster and more cost-effective than current methods 

In a recent study published in Science, researchers characterized a new family of effector proteins, named Cami1, using bioinformatics, biochemical and structural studies. They showed that when a virus attacks a bacterium, CRISPR-Cas10 signaling molecules activate Cami1, a ribosome-dependent ribonuclease. Activated Cami1 cleaves mRNAs involved in protein synthesis, inhibiting cell growth. This saves resources and prevents the production of viral proteins. Cami1's interaction with a specialized ribosomal structure, called a ribosomal stalk, is necessary for its entry into the protein synthesis center. Interestingly, the same capture mechanism to bind the ribosome is used by plant antiviral proteins that also inactivate ribosomes. This discovery has unveiled an additional layer of the CRISPR-Cas antiviral defense system and demonstrated a common antiviral strategy shared between eukaryotes and bacteria. Knowledge of our characterized Cami1 proteins will contribute to the development of new molecular tools in biotechnology and therapy

Solid tumors generate anti-immune signals that deactivate CAR T cells, making treatment less effective. To solve this problem, scientists have combined CAR T cells with cytokine injection, which can cause significant unintended toxicities. Researchers replaced the extracellular domain of various cytokine receptors with leucine zippers to create constitutively active receptors. CAR T cells expressing one of these chimeric cytokine receptors had superior antitumor activity against several types of cancer in cell lines and mouse models compared with conventional CAR T cells. Although chimeric cytokine receptors give a constant "on" signal to CAR T cells, they do not induce non-specific proliferation of CAR T cells. The system thus limits the effect of cytokine signaling to modified cells only, reduces the risk of cytokine-related toxicity, and provides a signal that these CAR T cells should function effectively in a suppressive tumor microenvironment. 

CRISPR/Cas9 method can lead to unintended DNA mutations that can have negative effects. Recently, Japanese researchers have developed a new gene-editing technique that is as effective as CRISPR/Cas9, yet significantly reduces these unintended mutations. In a new study published in Nature Communications , researchers led by Osaka University have introduced a new technique called NICER, based on the creation of several small cuts in single DNA strands by an enzyme  Cas 9 nickase. For their first experiments, the research team used human lymphoblastic cells with a known heterozygous mutation in a gene called TK1. When these cells were treated with nickase to induce a single cut in the TK1 region, TK1 activity was recovered at a low rate. However, when nickase induced multiple cuts in this region on both homologous chromosomes, the efficiency of gene correction was increased approximately seventeen-fold via activation of a cellular repair mechanism. Because the NICER method does not involve DNA double-strand breaks or the use of exogenous DNA, this technique appears to be a safe alternative to conventional CRISPR/Cas9 methods. 

Gene therapy that alters hemoglobin genes may be an answer to curing sickle cell disease (SCD) and beta thalassemia. These two common life-threatening anemias afflict millions of individuals across the globe. Scientists at St. Jude Children's Research Hospital and the Broad Institute of MIT and Harvard used a next-generation genome editing technology, adenosine base editing, to restart fetal hemoglobin expression in SCD patient cells. The approach raised the expression of fetal hemoglobin to higher, more stable, and more uniform levels than other genome editing technologies that use CRISPR/Cas9 nuclease in human hematopoietic stem cells. The findings were published in Nature Genetics. 

 A phase I clinical trial of an adjuvant personalized mRNA neoantigen vaccine, autogene cevumeran, in patients with pancreatic ductal carcinoma demonstrates that the vaccine can induce T cell activity that may correlate with delayed recurrence of disease.

Researchers are developing lipid nanoparticles that may target the lungs. The particles are made of molecules that contain two parts: a positively charged head group and a long lipid tail. The positive charge of the head group helps the particles interact with negatively charged mRNA, and also helps the mRNA escape from cellular structures that engulf the particles once they enter the cells. In tests on mice, the researchers showed that they could use the particles to deliver mRNA encoding CRISPR/Cas9 components designed to turn off a genetically encoded stop signal in the animals' lung cells. When this stop signal is removed, a gene for a fluorescent protein lights up. Measuring this fluorescent signal allows researchers to determine what percentage of cells have successfully expressed the mRNA. After one dose of mRNA, about 40% of lung epithelial cells were transfected, the researchers found. Two doses brought the level to more than 50% and three doses to 60%. The most important targets for treating lung disease are two types of epithelial cells called club cells and hair cells, and each was transfected at about 15%. These particles could offer an inhalable treatment for cystic fibrosis and other lung diseases. 

One of the challenges of CAR T cell therapy in solid tumors is a phenomenon known as T cell exhaustion. Previous studies have alluded to the inflammatory regulator Regnase-1 as a potential target to indirectly overcome the effects of T-cell exhaustion, as it can cause hyperinflammation when disrupted in T cells, reviving them to produce an antitumor response. The research team hypothesized that targeting the related but independent Roquin-1 regulator at the same time could boost responses further. The team used CRISPR-Cas9 gene editing to knock out Regnase-1 and Roquin-1 individually and together in healthy donor T cells with two different immune receptors that are currently being studied in Phase I clinical trials: the mesothelin-targeting M5 CAR (mesoCAR) and the NY-ESO-1-targeting 8F TCR (NYESO TCR). Following CRISPR editing, the T cells were expanded and infused into solid tumor mouse models, where the researchers observed that the double knockout resulted in at least a 10-fold increase in modified T cells compared to knocking down Regnase-1 alone, as well as increased anti-tumor immune activity and longevity of modified T cells. In some mice, this also led to an overproduction of lymphocytes, causing toxicity.

Although gene editing technologies hold promise for preventing and treating debilitating inherited diseases, a new study reveals limitations that must be overcome before gene editing to establish a pregnancy can be considered safe or effective. The study, published recently in the journal Nature Communications, involved sequencing the genomes of early human embryos that had undergone genome editing using the CRISPR gene editing tool. The work calls into question the accuracy of a DNA reading procedure that relies on amplifying a small amount of DNA for genetic testing. This scientific method commonly used to analyze a tiny amount of DNA in early human embryos fails to accurately reflect genetic changes. In addition, the study also reveals that gene editing to correct disease-causing mutations in early human embryos can also result in unintended and potentially harmful changes to the genome. The findings raise a new scientific basis for caution for any scientist who might be about to use genetically modified embryos to establish pregnancies.

Preimplantation genetic testing has become standard care for parents at risk of having children with chromosomal and single gene disorders. Embryos created via in vitro fertilization (IVF) can be genetically screened for abnormalities prior to implantation, thus minimizing the risk of inherited genetic disease. However, most human traits are highly polygenic. Preimplantation genetic testing for polygenic risk (PGT-P) is an emerging technology that can screen the entire genome of an embryo and uses polygenic indices to predict the likelihood of a particular polygenic phenotype occurring if used in IVF. Targeted outcomes range from risk of cancer and other diseases to a child's potential educational attainment. Although PGT-P is available in IVF clinics around the world, it remains unregulated in the United States and has received far less public attention and policy discussion than other technologies that seek to exert control over genetic traits, such as germline gene editing via CRISPR. To better understand the public's views toward PGT-P, researchers conducted a prerecorded national survey experiment on attitudes toward PGT-P. The authors found that moral acceptability and willingness to use PGT-P were higher than those for germline gene editing. 

The recent approval of a CRISPR-Cas9 therapy for sickle-cell anemia demonstrates that gene-editing tools can do an excellent job of eliminating genes to cure inherited diseases. But it is still not possible to insert entire genes into the human genome to replace them with defective or deleterious genes. A new technique, called RNA-mediated Precise Transgene Insertion, or PRINT, exploits the ability of certain retrotransposons to efficiently insert whole genes into the genome without affecting other genome functions. PRINT would complement the recognized ability of CRISPR-Cas technology to deactivate genes, perform point mutations and insert short segments of DNA. For PRINT, one piece of delivered RNA encodes a common retroelement protein called the R2 protein, which has several active parts, including a nickase and a reverse transcriptase. The other RNA is the template for the transgenic DNA to be inserted, as well as the elements controlling gene expression

Axi-cel CAR T therapy targets the CD19 molecule on large B-cell lymphoma cells. The ZUMA-7 trial demonstrated that axi-cel reduced the risk of disease progression, need for retreatment or death by 60% compared with standard therapy. Despite these positive results in terms of event-free survival and overall survival, some patients did not respond well to treatment or relapsed rapidly after treatment. Researchers analyzed tumor gene expression patterns from patient samples and determined that a B-cell gene expression signature and CD19 protein expression were significantly associated with improved event-free survival for patients treated with axi-cel but not with standard therapy. Patients with lower tumor cell levels of CD19 showed gene expression patterns associated with immunosuppression. These observations suggest that the tumor immune environment may play an important role in regulating axi-cel treatment and outcome. Furthermore, biomarkers associated with improved axi-cel treatment outcomes decreased as patients received more treatments, suggesting that receiving axi-cel as part of earlier treatment lines is essential to ensure better patient outcomes.

Genetic control in mammalian cells is essential for the development of safe and effective gene therapies. Current methods are associated with certain drawbacks, such as undesirable immunological responses, limited efficacy and overexpression of therapeutic genes. Current gene transfer technologies, such as adeno-associated viruses (AAVs), have difficulty achieving conditional and reversible gene control. Toxic ligands, leakage and high ligand concentrations, as well as small dynamic range are some of the limitations associated with current RNA-based systems. In a recent study researchers describe the pA regulatory system, inserted into cells by CRISPR-Cas9, based on a ribonucleic acid (RNA)-based switch to regulate mammalian gene expression by modulating the cleavage of a synthetic polyA signal (PAS) at a transgenic 5' untranslated region. (UTR). This technique differs from traditional riboswitch systems in that the PAS is present in the 5' UTR, combines the effects of numerous aptamers and uses two processes of Tc binding and alternative splicing. However, the new system can only use Tc as an inducing ligand, which cannot effectively penetrate all body tissues.

Using CRISPR screening, the deubiquitinase ATXN3 was identified as a key regulator of PD-L1 transcription in tumor cells, a critical factor in tumor immune evasion. In this study, researchers transfected a targeted library of all 96 members of the mammalian deubiquitinase family into melanoma cells, then sorted the cells according to low and high PD-L1 expression to identify the regulators. ATXN3 was found to positively influence PD-L1 transcription, helping tumor cells to evade the immune system. This new insight represents a promising target for improving the efficacy of antitumor immunotherapy by blocking checkpoints, potentially transforming cancer treatment strategies. The study also highlights the broader role of ATXN3 in regulating tumor microenvironmental responses to hypoxia and inflammation, opening up new avenues for cancer research and treatment.

Glioblastoma is the most common fatal brain cancer, and patients still lack good treatment options. Patients typically receive chemotherapy, radiotherapy and surgery, but most relapse within a few months. However, CRISPR looks very promising as a therapeutic strategy for the aggressive and difficult-to-treat brain cancer known as primary glioblastoma, according to the results of a new study from the Gladstone Institutes. Using computational methods to analyze entire cancer cell genomes, the team dove deep into non-coding DNA to identify the repetitive code they all shared, even if they harbored a different variety of mutations. The researchers then programmed CRISPR to focus on repetitive DNA sequences present only in recurrent tumor cells to destroy these cells. Working with cell lines from a patient whose glioblastoma had recurred after previous treatments, the team used CRISPR to destroy tumor cells while sparing healthy cells.

Severe combined immunodeficiency represents rare monogenic disorders with defects in humoral and cellular immunity. RAG genes code for proteins that initiate lymphoid-specific V(D)J recombination, essential for T and B lymphocyte maturation. As a result, individuals carrying pathogenic RAG variants are either completely devoid of, or possess far fewer, B and T cells. In a recent study, researchers described a CRISPR-Cas9-based genome-editing approach to replace the entire respective RAG2 coding sequences (CDS) with a corrective transgene in a 34-positive (CD34+) hematopoietic stem and progenitor cell (HSPC) differentiation cluster. Two recombinant adeno-associated virus serotype 6 (rAAV6) vectors were synthesized to integrate a green fluorescent protein expression cassette and a bovine growth hormone polyA sequence into CD34+ HSPCs after delivery of a single modified RAG2 guide RNA or the Cas9 ribonucleoprotein complex. The researchers' strategy completely replaced RAG2 CDS to treat autosomal recessive immunity disorders. The method induced expression of RAG2 coding DNA without exceeding endogenous RAG2 expression levels. Correction donors promoted the successful recombination and differentiation of V(D)J into CD3 + TCRγδ + and CD3 + TCRαβ + T cells. 

Fabry disease is caused by a genetic deficiency of α-galactosidase A (GLA), leading to the accumulation of its substrates such as globotriaosylceramide and globotriaosylsphingosine. Researchers have therefore developed a modified enzyme, modified α-N-acetylgalactosaminidase (mNAGA), to cure Fabry disease by changing the substrate specificity of NAGA to that of GLA. In this study, researchers tested whether genome-editing transplantation of mNAGA-secreting induced pluripotent stem cells (iPS) cells could deliver GLA activity in vivo. They therefore generated mNAGA-secreting iPS cells by TALEN-mediated knock-in at the AAVS1 site, a refuge locus. Furthermore, to exclude possible immunogenic reactions caused by endogenous GLA from iPS cells in patients, they disrupted the GLA gene by CRISPR-Cas9. When cardiomyocytes and fibroblasts from the Fabry model without GLA activity were co-cultured with mNAGA-secreting iPS cells, GLA activity was restored by mNAGA-expressing cells in vitro. Next, they transplanted the mNAGA-secreting iPS cells into the testes of mouse models of Fabry disease. After 7 or 8 weeks, GLA activity in the liver was significantly improved, although no recovery of activity was observed in the heart, kidneys or blood plasma.

Researchers report, in Nature Medicine, the interim results of a first-in-man phase 1 clinical trial evaluating the safety, antitumor activity and immunological characteristics of a genetically engineered natural killer (NKT) cell immunotherapy for neuroblastoma, a childhood tumor that most commonly arises in the adrenal gland. NKT cells were engineered to express a GD2-specific CAR, which enables immune cells to target a molecule found on the surface of neuroblastoma cells, and interleukin-15 a natural protein that supports NKT cell survival. Based on results obtained in 12 patients with recurrent stage 4 neuroblastoma resistant to other therapies, the researchers found that the treatment was safe for all 12 patients on four doses. No dose-limiting toxicities were reported. A further discovery revealed a regulatory gene in NKT cells that could have an impact on treatment efficacy. Leveraging the multiomics platform of key collaborator Immunai, Inc. the researchers discovered that up-regulation of the anti-proliferation factor 1 gene BTG in CAR NKT-infused cells indicates cell exhaustion and limits the functional activity of CAR NKT cells. Conversely, artificially reducing BTG1 expression in CAR NKT cells enhanced their therapeutic activity against neuroblastoma in a mouse model

One of the drawbacks of genome editing is that there are growing concerns about mutations and off-target effects. Researchers then hypothesized that current editing protocols that use Cas9 cause excessive DNA cleavage, resulting in some of the mutations. To test this hypothesis, the researchers built a system called "AIMS" in mouse cells, which assessed Cas9 activity separately for each chromosome. Their results showed that the commonly used method was associated with very high editing activity. They determined that this high activity caused some of the undesirable side effects, so they looked for gRNA editing methods that could suppress it. They found that an additional cytosine extension at the 5' end of the gRNA was effective as a "safeguard" against overactivity and controlled DNA cleavage. As a result of this study, the first mathematical model of the correlation between various genome editing patterns and Cas9 activity was created that can maximize the desired editing efficiency by developing activity-regulating gRNAs with appropriate Cas9 activity.

A new study shows that some effective cancer treatments, such as CAR-T cells, significantly improve quality of life six months after receiving therapy. To conduct the study, researchers recruited 103 patients aged 23 to 90 years with a diagnosis of blood cancer from April 2019 to November 2021. The researchers administered self-reported questionnaires measuring quality of life variables at time intervals including before CAR-T cell infusion and one week, one month, three months, and six months after CAR-T cell infusion. Quality of life was measured using a 27-item questionnaire known as the General Cancer Therapy Functional Assessment, which is composed of four different subscales (physical, functional, emotional, and social). Psychological distress was measured using the Hospital Anxiety and Depression Scale. Finally, major depressive symptoms were measured using the PHQ-9, and symptoms of posttraumatic stress disorder were measured using the Posttraumatic Stress Checklist. While most study participants eventually experienced an improvement in quality of life, approximately 20% of patients experienced persistent physical and psychological symptoms, which at times interfered with their quality of life.

Glaucoma is a neurodegenerative disease that causes vision loss and blindness due to a damaged optic nerve. Unfortunately, there is currently no cure. In a paper recently published in Communications Biology, researchers found that restoring mitochondrial homeostasis in diseased neurons can protect optic nerve cells from damage. The research team used induced pluripotent stem cells from glaucoma and non-glaucoma patients as well as clustered regularly spaced short palindromic repeats (CRISPRs) from human embryonic stem cells with glaucoma mutation. Using optic nerve stem cell differentiated retinal ganglion cells, electron microscopy and metabolic analysis, the researchers identified glaucomatous retinal ganglion cells with mitochondrial deficiency with a higher metabolic load on each mitochondrion, which leads to mitochondrial damage and degeneration. However, the process could be reversed by enhancing mitochondrial biogenesis with a pharmacological agent. The team showed that retinal ganglion cells are very efficient at degrading bad mitochondria, but at the same time produce more to maintain homeostasis.

Pancreatic cancer is an incurable form of cancer and gene therapies are currently in clinical trials to treat this deadly disease. A comprehensive review of gene and cell biotherapies in development to combat pancreatic cancer is published in the peer-reviewed journal Human Gene Therapy. The article, "Pancreatic Cancer Cell and Gene Biotherapies: Past, Present and Future," by corresponding author Pierre Cordelier of the University of Toulouse, and co-authors describes ongoing gene therapy clinical trials. In addition to gene therapies, the authors discuss vaccines, chimeric antigen receptor (CAR) T-cell therapy, suicide genes and oncolytic viruses.