Genetic Engineering - GEG Tech top picks
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PNAS | Mobile | ViroBlogy

PNAS | Mobile | ViroBlogy | Genetic Engineering - GEG Tech top picks | Scoop.it
In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus… ...
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New element about the RNA interference world!

 

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Antibiotic use, certain gut bacteria affect CAR-T efficacy, toxicity

Antibiotic use, certain gut bacteria affect CAR-T efficacy, toxicity | Genetic Engineering - GEG Tech top picks | Scoop.it
The use of broad-spectrum antibiotics in the month leading up to chimeric antigen receptor T-cell therapy led to poorer outcomes and increased treatment-related toxicities, study results showed.Certain types of gut bacteria had an impact on treatment efficacy and related toxicities among patients who received CAR-T for non-Hodgkin lymphoma or acute lymphoblastic leukemia, researchers reported.
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Researchers examined two cohorts of patients who received CD19-directed CAR-T cells for non-Hodgkin's lymphoma or B-cell acute lymphoblastic leukemia. The researchers used the first cohort to retrospectively examine the impact of prior antibiotic use on CAR-T clinical outcomes. The second cohort had prospective baseline fecal samples collected before CAR-T cell infusion for subsequent assessment of the fecal microbiome. The investigators determined that the use of certain antibiotics including piperacillin/tazobactam, meropenem, and imipenem/cilastatin, negatively affected OS (HR = 1.71; 95% CI, 1.12-2.59). The investigators also noted a significantly increased risk of immune effector cell-associated neurotoxicity syndrome among the entire cohort evaluated for pre-CAR-T antibiotic use (P = 0.023). Stool samples from the prospective cohort demonstrated an altered fecal microbiome including lower alpha diversity, increased frequency of bacterial dominance, and altered bacterial composition compared with healthy controls. The results of this study suggest that the composition of the gut microbiome affects CAR-T outcomes, but the investigators cautioned that further prospective studies in larger patient populations are needed to confirm the associations.

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Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities | Nature Communications

Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities | Nature Communications | Genetic Engineering - GEG Tech top picks | Scoop.it
The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9’s activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor’s activity. Screening combinatorial mutants is too massive for wet-lab experiment alone. Here the authors present a machine learning-coupled combinatorial mutagenesis approach to vastly reduce experimental burden for engineering Cas9 genome editing enzymes.
BigField GEG Tech's insight:

Staphylococcus aureus Cas9 (SaCas9) is an excellent candidate for in vivo gene therapy due to its small size allowing packaging into adeno-associated viral vectors for delivery into human cells for therapeutic applications. However, its gene editing activity may be insufficient for specific disease loci. The Cas9 protein contains several parts, including the protospacer-adjacent motif (PAM) interacting domain (PI) and Wedge (WED) to facilitate its interaction with the target DNA duplex. A research team therefore coupled machine learning and high-throughput screening platforms to design a SaCas9 protein with enhanced activity by combining mutations in its PI and WED domains surrounding the DNA duplex carrying a (PAM). PAM is essential for Cas9 to modify the target DNA and the idea was to reduce the PAM constraint for broader genome targeting while securing the protein structure by strengthening the interaction with the PAM-containing DNA duplex via the WED domain. The research team therefore designed new variants of Staphylococcus aureus Cas9 (SaCas9) with improved gene-editing efficiency, i.e. up to 33% improved activity at specific genomic loci The results are now published in Nature Communications and a patent application has been filed based on this work.   

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Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases - Cell

Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases - Cell | Genetic Engineering - GEG Tech top picks | Scoop.it
Development of a programmable mitochondrial genome editing tool that enables efficient
and accurate A-to-G conversions at diverse target sites in human mitochondrial genome.
BigField GEG Tech's insight:

Jin-Soo Kim's team has discovered how to edit the mitochondrial genome to cure mitochondrial diseases. The first step was to adapt the basic editing system to induce C-to-T substitutions in mtDNA in mice because gene editing systems, such as CRISPR-Cas9, cannot edit mtDNA. Now the Kim lab has taken the next step: inducing A-to-G substitutions in mtDNA. Kim's lab has adapted basic editing systems to edit the mitochondrial genome in mice, inducing pathogenic mutations in the ND5 gene via the C-to-T substitution. They did this using DddAtox which acts on double-stranded DNA and was originally used in mitochondrial cytosine base editing systems. Using their own genome-wide library of DNA-binding transcription activator-like effector proteins (TALEs), they created a system to induce C-to-T modifications by fusing the TALEs to split halves of DddAtox and an uracil glycosylase inhibitor. In this system, the TALE acts in the same way as a CRISPR sgRNA by directing the DddAtox enzyme to a target sequence. Now, the ability to also make A-to-G substitutions allows the theoretical correction of about 83% of pathogenic single nucleotide mutations in the mitochondrial genome. The implications of the study also extend to mitochondrial genetic variations related to cancer and diabetes.   

Florencer Edwine's curator insight, July 13, 1:17 PM

 

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Scooped by BigField GEG Tech
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Lifelong multilineage contribution by embryonic-born blood progenitors - Nature

Lifelong multilineage contribution by embryonic-born blood progenitors - Nature | Genetic Engineering - GEG Tech top picks | Scoop.it

Cliquez ici pour éditer le contenu

BigField GEG Tech's insight:

A groundbreaking study reveals that blood cells come from not just one type of mother cell, but two, which could have implications for blood cancers, bone marrow transplantation and immunology. Fernando Camargo, PhD, of the Stem Cell Program at Boston Children's Hospital, led the study, published in Nature on 15 June. Camargo's team applied a barcoding technique they developed several years ago. Using an enzyme called transposase, or CRISPR gene editing, they inserted unique genetic sequences into mouse embryonic cells in such a way that all the cells that descend from them also carry these sequences. This allowed the team to follow the emergence of all the different types of blood cells and their origins, right up to adulthood. The researchers are now testing whether these results also apply to humans. If so, the discovery of these cells, known as multipotent embryonic progenitor cells, could lead to the development of new treatments to boost the immune system of the elderly. They could also shed new light on blood cancers, especially in children, and help make bone marrow transplants more effective. 

Florencer Edwine's curator insight, July 13, 1:17 PM

 

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Researchers reveal important factor behind cancer therapy resistance and a way to counter it

Researchers reveal important factor behind cancer therapy resistance and a way to counter it | Genetic Engineering - GEG Tech top picks | Scoop.it
Why doesn't every cancer patient respond to immunotherapy? That's the question that researcher Daniel Peeper is looking to answer.
BigField GEG Tech's insight:

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.  

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Genetically engineered immune cells target cancer cells that may be responsible for leukemia relapse

Genetically engineered immune cells target cancer cells that may be responsible for leukemia relapse | Genetic Engineering - GEG Tech top picks | Scoop.it
Genetically engineered immune cells successfully target the specific cancer cells that may be responsible for relapse of acute myeloid leukemia (AML), a type of blood cancer, and proved effective in animal models of the disease, according to a preclinical study by investigators at Weill Cornell Medicine.
BigField GEG Tech's insight:

Genetically modified immune cells successfully target specific cancer cells that may be responsible for the relapse of acute myeloid leukemia (AML). In a study published on 28 April in Nature Communications, the researchers developed a CAR T cell therapy (UCART123) targeting CD123, which is found on leukemia stem cells and enables T cells to seek out and attack cancer cells. When the team tested the UCART123 cells in a mouse model of AML, they found that the therapy effectively eliminated leukemia cells and prolonged survival. The scientists also devised a highly sensitive monitoring strategy to detect any residual cancer cells and assess the persistence of UCART123 cells. Finally, they demonstrated that UCART123 cells have specificity against leukemia cells, with minimal toxicity to normal blood cells in mice. The preclinical results led to a Phase 1 clinical trial testing UCART123 in patients with relapsed/refractory AML at several sites across the US, including New York-Presbyterian/Weill Cornell Medical Center. The results of the preclinical study suggest that UCART123 cells are highly selective and specific in targeting AML. 

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Multiplexed genome regulation in vivo with hyper-efficient Cas12a | Nature Cell Biology

Multiplexed genome regulation in vivo with hyper-efficient Cas12a | Nature Cell Biology | Genetic Engineering - GEG Tech top picks | Scoop.it
Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR–Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications. Through structure-guided protein engineering, Guo et al. developed a Lachnospiraceae bacterium Cas12a variant with enhanced editing efficiency and applied the enzyme-dead version of this variant for multiplex gene activation in the mouse retina.
BigField GEG Tech's insight:

The research group has designed a new Cas12a protein with extensive capabilities. In Nature Cell Biology, the team describes how the new hyperdCas12a variant can regulate the expression of several genes at once with great efficiency, including gene activation and repression. With promising results of multiplexed gene activation in mouse retinas, hyperdCas12a has many interesting potential applications, including in therapeutics. To demonstrate the potency of hyperdCas12a in regulating multiplexed genes, the team first tested it in mouse P19 cells derived from an embryonic teratocarcinoma. The team achieved simultaneous activation of the endogenous transcription factors Sox2, Klf4 and Oct4 using a single lncRNA array driven by the well-established U6 promoter, which is commonly used in CRISPR experiments due to its ability to drive sgRNA expression. The activation results obtained with hyperdCas12a were significantly higher than wild-type dCas12a: in the case of Klf4, there was a 70-fold increase in gene activation. The final test of the hyperdCas12a system was to synergistically co-activate the Sox2, Klf4 and Oct4 genes in vivo in mouse retinas. Researchers continue to adapt and improve the hyperdCas12a system, including directing the technology towards clinical applications. 

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Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells | Nature Biotechnology

Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells | Nature Biotechnology | Genetic Engineering - GEG Tech top picks | Scoop.it

Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that might lead to cell products with heterogeneous composition. Here we describe an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, after subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy. Implantable scaffolds rapidly generate and release anti-tumor CAR-T cells in mice.

BigField GEG Tech's insight:

Many people are excluded from CAR T cell-based treatments because of its cost. One reason for the high cost is that the manufacturing process is complex, time-consuming and must be individually tailored to each cancer patient. So to address this challenge, the researchers created a biotechnology called Multifunctional Alginate Scaffolds for T cell Engineering and Release (MASTER) that is a biocompatible sponge-like material. To begin treatment, the researchers isolate the patient's T cells and mix these naive T cells with the modified virus. The researchers pour this mixture onto MASTER, which absorbs it. MASTER is decorated with the antibodies that activate the T cells, so the cell activation process begins almost immediately. Meanwhile, MASTER is surgically implanted into the patient. After implantation, as the T cells become activated, they begin to respond to the modified viruses, which reprogram them into CAR-T cells. MASTER is also imbued with interleukin factors that promote cell proliferation. After implantation, these interleukins begin to leach out, promoting rapid proliferation of CAR-T cells. In a proof-of-concept study involving lymphoma in mice, researchers found that this treatment was faster and more effective than conventional CAR-T cell cancer treatment. 

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Researchers develop new precision medicine technology for cancer immunotherapy

Researchers develop new precision medicine technology for cancer immunotherapy | Genetic Engineering - GEG Tech top picks | Scoop.it
In recent years, great advances have been made in the development of new successful immunotherapies to treat cancer. CAR T-cell therapy and antibody treatments are two types of targeted immunotherapies that have revolutionized areas of cancer care.
BigField GEG Tech's insight:

CAR T-cell therapy and antibody treatments are two types of targeted immunotherapies that have revolutionized the fields of cancer care. However, there are still significant challenges in identifying cancer cell surface proteins as targets for immunotherapies. A research group at Lund University in Sweden is well on their way as they have developed a new precision medicine technology that allows for comprehensive mapping of the entire tumor cell surface antigen landscape in patients. The method developed by the research team, "Tumor Surfaceome Mapping, TS-MAP," allows direct analysis of all accessible tumor cell surface antigens in patient tumor tissue. In a close collaboration between neurosurgery, oncology and advanced proteomics in Lund, the researchers were able to identify several tumor cell surface antigens in fresh tissue from patients with aggressive brain tumors for which there is currently no effective treatment. An important advantage of the TS-MAP technology is that it provides a complete picture of the cell surface antigens displayed on the surface of the cancer cell, as well as information about specific cell surface antigens that have a high capacity to infiltrate cancer cells, and can destroy them from within. 

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CRISPR activation and interference screens decode stimulation responses in primary human T cells - Science

CRISPR activation and interference screens decode stimulation responses in primary human T cells - Science | Genetic Engineering - GEG Tech top picks | Scoop.it
Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators require
BigField GEG Tech's insight:

CRISPR genome editing has served as a powerful tool to delete or modify DNA sequences and study the resulting effect. Now, researchers at the Gladstone Institutes and UC San Francisco (UCSF) have co-opted the CRISPR-Cas9 system to forcibly turn on genes rather than edit them in human immune cells. The method, known as CRISPRa, allowed them to discover genes that play a role in immune cell biology more thoroughly and quickly than before. The study, published in the journal Science, is the first to successfully use CRISPRa on a large scale in primary human cells, which are cells isolated directly from a person. In the new work, Marson, Steinhart and co-first author Ralf Schmidt, MD, worked with their colleagues to adapt CRISPRa and CRISPRi to work at high efficiency in primary T cells, something never done before. Improving the efficiency of delivery of the CRISPRa or CRISPRi machinery into cells was essential to enable genome-wide experiments and accelerate discovery. Marson's lab is currently studying some of the individual genes identified in their screen, and working to further leverage CRISPRa and CRISPRi to discover genes that control other critical traits in human immune cells.  

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A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers | Nature Communications

A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers | Nature Communications | Genetic Engineering - GEG Tech top picks | Scoop.it
B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers. Antigen escape represents a potential drawback of chimeric antigen receptor T cell (CAR-T) therapy targeting a single tumor-associated antigen. To reduce the risk of antigen escape, here the authors report the design and characterization of a BAFF ligand CAR-T that can recognize three different receptors (BAFF-R, BCMA and TACI), demonstrating in vitro and in vivo cytotoxicity against multiple B cell cancer models.
BigField GEG Tech's insight:

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. 

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CAR T cells produced in vivo to treat cardiac injury - Science

CAR T cells produced in vivo to treat cardiac injury - Science | Genetic Engineering - GEG Tech top picks | Scoop.it
Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by deliverin
BigField GEG Tech's insight:

The standard CAR T cell strategy would be problematic when directed against heart failure or other fibrotic diseases in humans.

Fibroblasts have a normal and important function in the body, particularly in wound healing. CAR T cells that are genetically reprogrammed to attack fibroblasts could survive in the body for months or even years, suppressing the fibroblast population and impairing wound healing for all that time. Therefore, in the new study published in Science, Epstein and colleagues designed a technique for a more temporary and controllable, and much more procedurally simple, type of CAR T cell therapy. They designed an mRNA that encodes a T-cell receptor targeting activated fibroblasts and encapsulated the mRNA in tiny bubble-like lipid nanoparticles, which are themselves coated with molecules that lodge in T cells. Injections of this therapy into mice modeling heart failure successfully reprogrammed a large population of mouse T cells, causing a major reduction in cardiac fibrosis in the animals and restoration of a mostly normal heart size and function with no sign of continued anti-fibroblast T cell activity one week after treatment. Researchers continue to test this mRNA-based transient CAR T-cell technology, with the hope of eventually starting clinical trials

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Off-the-shelf, gene-edited CAR-T cells forge ahead, despite safety scare - Nature

Off-the-shelf, gene-edited CAR-T cells forge ahead, despite safety scare - Nature | Genetic Engineering - GEG Tech top picks | Scoop.it
Race to the clinic reignites for an off-the-shelf alternative to autologous CAR-T cell therapy, even as concerns over chromosomal abnormalities linger.
BigField GEG Tech's insight:

The race to the clinic is reviving for a ready-made alternative to autologous CAR-T cell therapy, even as concerns about chromosomal abnormalities persist. The Advanced Regenerative Medicine Therapy designation, which makes the therapy eligible for accelerated approval, will also help remove a veil that has hung over standard CAR-T cell therapies since October, when the FDA put all trials of competitor Allogene Therapeutics on hold following the detection of a chromosomal abnormality in a patient who received ALLO-501A in a Phase 2 trial. The FDA's green light for CRISPR Therapeutics dispels broader concerns that the agency views this type of genotoxic safety event as an intractable problem for the entire class of allogeneic CAR-T therapies. Today, many companies are eliminating loci associated with the MHC-I to avoid host T cell recognition of transplanted CAR-T cells. Companies also equip their T cells with a variety of safety switches and performance enhancers.

However, as the complexity of the assembly increases, the risk of off-target effects also increases. This may be important from a safety perspective, given that most cancers lack unique antigens. Achieving rapid remission and re-dosing if necessary, can minimize the toxic effects that CAR-T cells can have on healthy tissues expressing the targeted antigen.

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The identification and characterization of a thermophilic Cas13a ortholog highly active at the temperatures required for RT-LAMP

The identification and characterization of a thermophilic Cas13a ortholog highly active at the temperatures required for RT-LAMP | Genetic Engineering - GEG Tech top picks | Scoop.it
Researchers developed a new detection assay for SARS-CoV-2.
BigField GEG Tech's insight:

In a recent study published in Proceedings of the National Academy of Sciences, researchers developed a new assay for the detection of SARS-CoV-2. For the test, CRISPR systems were coupled to loop-mediated isothermal amplification (LAMP) or recombinase polymerase (RPA) amplification to boost sensitivity and improve detection at attomolar levels. In addition, reverse transcription (RT)-RPA/LAMP systems coupled with Cas13 systems have been developed for the detection of SARS-CoV-2. RT-LAMP has become the preferred method due to its high sensitivity, low cost and ease of use. Since these systems use functional Cas enzymes at 37°C and LAMP requires high temperatures (55°C to 65°C), the RT-LAMP/RPA assays coupled with CRISPR. The multiplexed assay therefore has a thermophilic Cas13a isolated from Thermoclostridium caenicola (TccCas13a) and uses it for the detection of SARS-CoV-2 using fluorescent amidite-labelled RNA and has developed an AapCas12b-based RNase P detection using HEX-labelled ssDNA reporters. The researchers achieved simultaneous detection of RNase P and SARS-CoV-2 without any interference from the fluorescence signals. A mobile phone application was developed to collect and interpret the fluorescence viewer readings at low cost. 

 

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Preclinical study unlocks the promise of CAR T-cell therapy for treatment of solid tumors

Preclinical study unlocks the promise of CAR T-cell therapy for treatment of solid tumors | Genetic Engineering - GEG Tech top picks | Scoop.it
Scientists at St. Jude Children's Research Hospital identified a molecular mechanism that in a preclinical study unlocked the promise of CAR T–cell therapy for treatment of solid tumors.
BigField GEG Tech's insight:

Currently, too few CAR T cells become memory cells that persist and create more T cells in the long term. However, a group of researchers recently showed that the distribution of the c-Myc protein in a parental T cell may be important for this process and published this work in the journal Nature. The researchers knew that a daughter cell with more c-Myc became an effector cell. In this study, the team found that the protein complex cBAF (canonical Brg1/Brg-associated factor) interacted with c-Myc. Daughter cells with high concentrations of cBAF and c-Myc became effector T cells. The cBAF binds certain regions of chromatin, proteins on DNA. The discovery suggests that it can guide the fate of cells, what type of T cells they become, by controlling the expression of effector cell-related genes. The distribution of cBAF occurs in the first activated T cell that begins the adaptive immune response; therefore, the researchers realized that cell fate is decided early in the immune response. The researchers used the molecular information they discovered. They applied a cBAF inhibitor during CAR T cell activation to generate more memory T cells. In a preclinical model, T cells treated with an inhibitor-controlled tumor growth better than untreated cells. The treated cells also survived longer and in greater numbers.

Florencer Edwine's curator insight, July 13, 1:15 PM

 

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In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice | Nature Biotechnology

In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice | Nature Biotechnology | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

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. 

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Potentiating adoptive cell therapy using synthetic IL-9 receptors

Potentiating adoptive cell therapy using synthetic IL-9 receptors | Genetic Engineering - GEG Tech top picks | Scoop.it
Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD–IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours. Synthetic chimeric orthogonal IL-2 receptors that incorporate the intracellular domain of receptors for other γ-chain cytokines such as IL-9 can reroute orthogonal signalling and alter the phenotype of T cells to improve anti-tumour responses.
BigField GEG Tech's insight:

Researchers have shown that a synthetic IL-9 receptor allows anti-cancer T cells to do their job without the need for chemo or radiation. T cells modified with the synthetic IL-9 receptor were potent against tumours in mice, as published in Nature. This group of researchers were interested in testing modified versions of the synthetic receptor that transmit other cytokine signals from the common gamma chain family: IL-4, -7, -9 and -21. Of the synthetic common gamma chain signals, the IL-9 signal was worth studying and unlike other cytokines, IL-9 signalling is not generally active in naturally ocurring T cells. The synthetic IL-9 signal gave the T cells a unique blend of stem cell and killer cell qualities that made them more robust in fighting tumours. In particular, the researchers targeted two types of difficult-to-treat cancer models in mice: pancreatic cancer and melanoma. They used T cells targeted to the cancer cells via the natural T cell receptor or a chimeric antigen receptor (CAR). In all cases, T cells engineered with synthetic IL-9 receptor signalling were superior and helped cure some tumours in mice when they could not do otherwise. 

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CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context | Nature Communications

CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context | Nature Communications | Genetic Engineering - GEG Tech top picks | Scoop.it
A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 “sliding” on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design. CRISPR/Cas9-mediated cleavage efficiency varies at different target locations. Here the authors explain this variation with binding free energy changes and show that overlapping Cas9 binding sites influence cleavage efficiency by enabling Cas9 sliding.
BigField GEG Tech's insight:

A major drawback of CRISPR-Cas9-mediated genome editing is that not all guide RNAs (gRNAs) efficiently cleave target DNA. Although the heterogeneity of gRNA activity is well recognized, the exact way in which CRISPR-Cas9 activity is regulated is not fully understood. In a recent study, a team of researchers used an energy-based model to identify the mechanisms regulating CRISPR-Cas9 activity and specificity. The team investigated the binding free energy, Δ G H , of RNA-DNA interactions and the effects of energy changes during RNA-DNA hybridisation on the efficiency of 11,602 Cas9 gRNAs experimentally validated in other studies. The efficiency of the gRNAs was measured as indel frequency, which is the most accurate indicator of CRISPR-Cas9 activity. They found that Cas9 'slippage' or binding competition on adjacent overlapping protospacer motifs (PAMs) affected gRNA activity by regulating local RNA-DNA interactions. Furthermore, the binding free energy sweet spot quantitatively defines the optimal activity of CRISPR-Cas9 at target and off-target sites in that the gRNA does not bind too weakly or too strongly. In the future, the team will continue to improve their methods, which will include further optimization of the gRNA design for increased efficiency and specificity. 

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