Genetic Engineering Publications - GEG Tech top picks

An evolution of the CRISPR-Cas9 gene editing technology, master editing has enormous potential to treat genetic diseases in humans. However, to date, the factors determining successful editing are not well understood. A new tool to predict the chances of successfully inserting a gene-edited DNA sequence into the genome of a cell has been developed by researchers. The study, published in Nature Biotechnology, evaluated thousands of different DNA sequences inserted into the genome using master editors. This data was then used to train a machine learning algorithm to help researchers design the best solution for a given genetic defect, promising to speed up efforts to introduce master editing into the clinic. Sequence length proved to be a key factor, as was the type of DNA repair mechanism involved. The next steps for the team will be to create models for all known human genetic diseases to better understand if and how they can be corrected using master editing.

A group of researchers set out to identify the genes that play a role in immunotherapy resistance. In today's cover story in Cell Reports Medicine, they describe an important factor behind this resistance to therapy and a potential way to counter it. The researchers grew tumour cells in their laboratory and inactivated one gene per cell at a time using the CRISPR/Cas9 technique. Then they treated the tumour cells with T cells or NK cells to analyse which genes were involved in resistance against the immune cells. This led to the discovery of three genes from the same family. The screening led us to an entire gene family, which is a real success. Indeed, when they switched off these genes including RNF31, the tumour cells were destroyed much more efficiently by T cells and NK cells. In addition, they found that inhibiting RNF31 also increased the sensitivity of T cells to tumour cells that were invisible on the surface of immune cells. This so-called bystander effect may amplify the effect of the treatment.  

Researchers at Seidman Cancer Center and Case Western Reserve University Hospitals have developed a new approach to CAR T cell therapy for B-cell cancers that triples targeted antigens on cancer cells. This approach promises to significantly reduce the potential for antigen escape currently found in CAR T therapies that target only CD19. The novel B-cell activating factor (BAFF) CAR T product developed here specifically binds to each of three receptors instead of one - BAFF-R, BCMA and TACI, providing more therapeutic options. At least two of these three receptors are found in almost all B-cell cancers, with some cancers expressing all three. Experimental results reported in Nature Communications show that BAFF CAR T is effective in killing several B-cell cancers. In addition, studies show robust in vitro and in vivo cytotoxicity exerted by CAR T BAFFs against mantle cell lymphoma, multiple myeloma, and mouse xenograft models of acute lymphoblastic leukemia. An Investigational New Drug application with the U.S. Food and Drug Administration will be filed in the coming weeks with Luminary Therapeutics and the team plans to initiate a clinical trial of BAFF CAR T therapy in patients with non-Hodgkin's lymphoma within the next few months. 

CAR T cell therapies directed against CD19 have demonstrated high rates of pre-malignant responses in patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL). However, long-term data on these therapies are limited. However, according to the results of a long-term follow-up analysis of a phase 1 study NCT01593696 published in the Journal of Clinical Oncology, sequential CAR-T cell therapy directed against CD19.28ζ followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) induces durable disease control in a significant population of pediatric and young adult patients with B-cell ALL. In the Phase 1 clinical trial, two doses of CAR T cell-based therapies were used, either an infusion of 1 x 10^6 CAR T cells/ kg administered on day 0, or an infusion of 3 x 10^6 CAR T cells/ kg administered on day 0. The maximum tolerated dose was defined as 1 x 10^6 CAR T cells / kg. Overall, 53 patients were enrolled in the study; 51 patients had B-ALL and 2 had diffuse large B-cell lymphoma. Complete responses were observed in 62% (n=31) of patients infused with CD19.28ζ directed CAR T cells (n=50).

The development of CAR T cells has facilitated the treatment of blood tumours. Furthermore, this therapy is not effective against solid tumours such as neuroblastoma and has even revealed toxic effects which are due to the fact that most of the antigens that cancerous tissue has on its surface are also found in healthy tissue. However, a group of scientists at Los Angeles Children's Hospital has developed a modified version of CAR T cells that looks promising for targeting neuroblastoma based on the pre-clinical phases. Their study was published in Nature Communications. The researchers used a new CAR T technology called Synthetic Notch (synNotch). SynNotch CAR T cells have a unique property. The special synNotch protein is designed to recognize the GD2 antigen. When it does, this protein instructs the cell to activate its CAR T properties, allowing it to recognize a second antigen: B7H3. By following these specific instructions, cells can only kill cells with both antigens and therefore mostly cancer cells. This triggering property minimizes toxicity because healthy cells will sometimes have low levels of one of the antigens but never both.  

https://www.nature.com/articles/s41467-020-20785-x

The new approach opens up nearly 90 percent of CRISPR-Cas systems for use in human cells, including biomedical research and potential gene and cell therapies.

In a study appearing on Sept. 23 in Nature Biotechnology, Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, and Adrian Oliver, a post-doctoral fellow in the Gersbach lab who led the project, describe how they successfully harnessed Class 1 CRISPR systems to turn target genes on and off and edit the epigenome in human cells for the first time.

A new technique will help biologists tinker with genes, whether the goal is to turn cells into tiny factories churning out medicines, modify crops to grow with limited water or study the effects of a gene on human health.

Garst et al. sought to combine these strategies to engineer precise designed mutations on a genomic scale. They generated a library of >50,000 plasmids containing 3 variable but covalently coupled components: a gRNA-expressing region, a replacement cassette to generate a defined mutation at the particular gRNA target site, and a barcode sequence to uniquely identify the overall construct. As an additional feature, the replacement cassette was designed to generate the main target-site mutation, as well as a synonymous mutation in a nearby protospacer-adjacent motif (PAM) to minimize further Cas9 cleavage at edited loci.

In initial proof-of-principle tests, the team transfected single constructs or pooled libraries of constructs into Cas9-expressing Escherichia coli cells and achieved ~70% efficiency of correct editing at target loci. They then tested single-gene CREATE libraries for functional screening: mutagenesis of folA (which encodes dihydrofolate reductase) to screen for trimethoprim resistance and mutagenesis of the acrB drug efflux pump to screen for isobutanol resistance. In both cases, sequencing across the barcode region in populations of E. coli cells was used to infer genome edits that were relatively enriched in the treated samples, and follow-up validation experiments using individual constructs confirmed that these edits indeed conferred treatment resistance.

The authors review on the present regulation of CRISPR and discuss the translational aspect of genome engineering research and patient autonomy with respect to the “right to try” potential novel non-germline gene therapies.

A new study from Tel Aviv University proposes a new and unique AIDS treatment that could be developed into a vaccine or a one-time treatment for HIV patients. The study examined the engineering of B-type white blood cells in the patient's body to secrete anti-HIV antibodies in response to the virus. The technique developed in his laboratory uses B-white blood cells that would be genetically modified inside the patient's body to secrete neutralizing antibodies against the HIV virus. The gene editing was done with a CRISPR system. The researchers are able to engineer the B cells inside the patient's body using two viral vectors from the AAV family, one encodes the desired antibody and the second encodes the CRISPR system. When CRISPR cuts the desired site in the genome of the B cells it directs the introduction of the desired gene: the gene coding for the antibody against the HIV virus. On the basis of this study, we can hope that in the next few years we will be able to produce a drug against AIDS in this way, but also against other infectious diseases, for certain types of cancer caused by a virus, such as cervical cancer. 

A collaborative study led by the Monash Biomedicine Discovery Institute has discovered a new immune checkpoint that could be exploited for cancer treatment. The study shows that by inhibiting the protein tyrosine phosphatase PTP1B in T cells, the body's immune response to cancer can be mobilized, helping to suppress tumor growth. Indeed, this study showed that using a new drug candidate, the abundance of PTP1B in tumor-infiltrating T cells is increased, limiting the ability of T cells to attack tumor cells and fight cancer. These findings identified PTP1B as an intracellular brake, or checkpoint, reminiscent of the PD-1 cell surface checkpoint whose blockade has revolutionized cancer treatment. Furthermore, beyond the improved response to PD-1 blockade, the authors showed that inhibition of PTP1B also significantly improved the efficacy of cell-based therapies using CAR T cells. The authors demonstrate that deletion or inhibition of PTP1B can significantly improve the ability of CAR T cells to attack solid tumors in mice, including breast cancer.  

MB-106 is a fully humanized, autologous, modified CAR T cell therapy that targets the CD20 protein on the surface of cancer cells. The researchers based the new agent on work done at the Fred Hutchinson Cancer Research Center, which is now collaborating with Mustang Bio to develop the cell therapy. MB-106 differs from approved commercial CAR T cell therapies in that it contains both the CD28 and 4-1BB costimulatory domains. Researchers modified its manufacturing process in 2019 to combine CD4-positive and CD8-positive cell culture for the final infusion product. Resarch team conducted a single-center phase 1/phase 2 dose escalation study to evaluate the safety and efficacy of MB-106 in patients with relapsed or refractory CD20-positive B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Nineteen of 20 patients (95%) achieved a response to a single infusion of MB-106. Twelve patients (65%) achieved a complete response. The CD20-directed CAR-T in this study will be better tolerated and may serve as an alternative for patients whose disease does not express the CD19 antigen.

Treatments based on immunotherapy give hope to many people with cancer that they will finally be cured. However, some treatments can be very expensive, have side effects, or may only work on a small number of people. That's why researchers at the University of Chicago's Pritzer School of Molecular Engineering have developed a new therapeutic vaccine using a patient's own tumor cells that have been modified to secrete vascular endothelial growth factor and then irradiated so that the cells are dead before being reinjected. This vaccine would therefore train the patient's immune system to detect and eradicate cancer because, according to clinical trials, it stops the growth of melanoma tumors in mouse models. In addition, an immunological memory is set up thanks to the vaccine and leads to long-term effects because the vaccine would destroy the appearance of new tumor cells 10 months after the injection. The injection of the vaccine is done like a traditional vaccine. The advantages of this vaccine are that it would be more effective, less expensive and much safer.

The network of blood vessels in the tumour microenvironment remains one of the most difficult blockages for cell therapies to penetrate and treat solid tumours. However, a new study published in Nature Cancer explains that researchers at Penn Medicine found that combining CAR T cell therapy with a PAK4 inhibitor drug allowed modified cells to work their way through and attack the tumour, leading to significantly improved survival in mice. The genetic reprogramming of tumour endothelial cells lining the walls of blood vessels is caused by an enzyme known as PAK4. Penn's team discovered that PAK4 inhibition reduces abnormal tumour vascularization and improves T cell infiltration and CAR T cell immunotherapies in mouse models of glioblastoma. An experiment with T-RCA cell therapy led by EGFRvIII and a PAK4 inhibitor showed a nearly 80 percent reduction in tumour growth compared to mice that received CAR T cell therapy only five days after infusion. The targeting of PAK4 may therefore provide a unique opportunity to recondition the tumour microenvironment and improve T-cell-based cancer immunotherapy for solid tumours.   

Here, scientists describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. They generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis.

In this chapter, the authors discuss the current state of epigenomic profiling and how functional information can be indirectly inferred. They also present new approaches that promise definitive functional answers, which are collectively referred to as 'epigenome editing'. In particular, they explore CRISPR-based technologies for single-locus and multi-locus manipulation. Finally, they discuss which level of function can be achieved with each approach and introduce emerging strategies for high-throughput progression from profiles to function.

The CRISPR/Cas9 gene editing method has been successfully applied to modify genomes in many organisms. However, several critical issues remain unresolved and have become major hurdles for its broad applications.