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Researchers discover a ‘weak spot’ that makes multi-drug resistant tumors vulnerable

Researchers discover a ‘weak spot’ that makes multi-drug resistant tumors vulnerable | Genetic Engineering - GEG Tech top picks | Scoop.it
One of the greatest challenges facing cancer researchers is to understand why some patients don't respond to treatments.
BigField GEG Tech's insight:

One of the biggest challenges facing cancer researchers is understanding why some patients do not respond to treatment. In some cases, tumours have what is known as multi-drug resistance (MDR), which severely limits treatment options for patients. However, researchers have discovered one of the causes of MDR and a potential strategy to combat it. In their study, the researchers began by using CRISPR technology in mouse stem cells to search for mutations that generate resistance to antitumor agents such as cisplatin, rigosertib or ultraviolet light. Mutations in the FBXW7 gene appeared early on, suggesting that this mutation could confer MDR. In addition, FBXW7-deficient cells showed an excess of mitochondrial proteins, which has already been shown to be associated with drug resistance. Nevertheless, a detailed analysis of these organelles further revealed that the mitochondria of these multi-drug resistant cells appeared to be under severe stress. Finally, the researchers showed that the antibiotic tigecycline is toxic to FBXW7-deficient cells because it activates the 'integrated stress response'. This study thus opens a new avenue of research to combat multidrug resistance.

 

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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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PD-1 Blockade in Mismatch Repair–Deficient, Locally Advanced Rectal Cancer | NEJM

PD-1 Blockade in Mismatch Repair–Deficient, Locally Advanced Rectal Cancer | NEJM | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

Breakthrough findings were presented at the 2022 ASCO Annual Meeting and published in The New England Journal of Medicine by researchers at Memorial Sloan Kettering Cancer Center (MSK) confirming a clinical complete response in all 14 patients who received the immunotherapy treatment dostarlimab as a first-line treatment for mismatch repair-deficient (MMRd) locally advanced rectal cancer. This new approach of “immunoablative” therapy uses immunotherapy to replace surgery, chemotherapy and radiation to remove cancer.

 

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Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells | Gene Therapy

Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells | Gene Therapy | Genetic Engineering - GEG Tech top picks | Scoop.it

We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.

BigField GEG Tech's insight:

 There is a pervasive sense of hopelessness in these non-small cell lung cancer (NSCLC) patients due to the current lack of treatment options, treatment failure due to drug resistance, and poor survival rates. Chemotherapy resistance is a major challenge in the treatment of NSCLC. Nuclear factor erythroid 2-related factor 2 (NRF2) may be at the root of this resistance. It is a master regulator of hundreds of other genes involved in numerous cytoprotective and metabolic pathways. Under normal physiological conditions, NRF2 protects cells from oxidative stress, toxic attacks and chemotherapy, keeping cells in homeostasis. Cancer cells hijack the NRF2 pathway and disrupt this function. When NRF2 becomes over-expressed and accumulates in the cell, the cell is able to fight off these toxic insults. This is really where the cancer becomes chemoresistant. Advanced patients have much higher levels of NRF2. However, in a paper published last month in Gene Therapy, a research team describes a new anti-cancer strategy that uses CRISPR-Cas9 to eliminate NRF2. The key finding of the study is that CRISPR-based disruption of NRF2 can lead to exon skipping. A dual sgRNA approach to target exon 4 resulted in the deletion of a 103-base-pair fragment, including exonic splice enhancer sequences. 

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Viable offspring derived from single unfertilized mammalian oocytes

Viable offspring derived from single unfertilized mammalian oocytes | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

In mammals, parthenogenesis is limited because of problems arising from genomic imprinting. Here, the scientists report live mammalian offspring derived from single unfertilized eggs. This was achieved by the targeted DNA methylation rewriting of seven imprinting control regions. By designing guide RNAs with protospacer adjacent motif (PAM) sequences matching one allele but not the other, dCas9-Dnmt3a or dCpf1-Tet1 enables targeted DNA methylation editing in an allele-specific manner. The success of parthenogenesis in mammals opens many opportunities in agriculture, research, and medicine.

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Study identifies new immune checkpoint that may be exploited for cancer treatment

Study identifies new immune checkpoint that may be exploited for cancer treatment | Genetic Engineering - GEG Tech top picks | Scoop.it
A collaborative study led by the Monash Biomedicine Discovery Institute has discovered a new immune checkpoint that may be exploited for cancer therapy.
BigField GEG Tech's insight:

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.  

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Case study highlights Parkinson’s-like toxicity after BCMA-directed CAR T-cell therapy

Case study highlights Parkinson’s-like toxicity after BCMA-directed CAR T-cell therapy | Genetic Engineering - GEG Tech top picks | Scoop.it
Clinicians who treat patients with chimeric antigen receptor T cells have become adept at identifying and treating acute neurotoxicity, a common adverse event associated with the therapy.Researchers from Mount Sinai published a case study in Nature Medicine about a patient who developed neurocognitive and hypokinetic movement disorder with features of Parkinson’s disease after receiving
BigField GEG Tech's insight:

Clinicians treating patients with CAR T cells have become adept at identifying and treating acute neurotoxicity, a common adverse event associated with the therapy. Researchers at Mount Sinai published a case study in Nature Medicine of a patient who developed a neurocognitive, hypokinetic movement disorder with features of Parkinson's disease after receiving the CAR-T therapy targeting the B-cell maturation antigen (BCMA) called ciltacabtagene autoleucel in the CARTITUDE-1 clinical trial. Since reports of this toxicity have not been observed with CD19-directed CAR-T therapies for other blood cancers, it appears to be specifically associated with BCMA-directed CAR-T. As it turns out, Parekh and colleagues found that BCMA is expressed on certain brain cells, and they saw evidence that BCMA-targeted CAR-T cells entered the patient's cerebrospinal fluid and crossed the blood-brain barrier. With ciltacabtagene autoleucel poised to become the second BCMA-directed CAR-T to gain commercial approval later this year, clinicians should discuss the potential benefits and risks of the therapy with their patients. They also need to be vigilant about screening for late neurological toxicities until researchers can make improvements to the new CAR-T constructs to mitigate the risk. 

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Safety an ‘important differentiating factor‘ for novel CAR-T in B-cell malignancies

Safety an ‘important differentiating factor‘ for novel CAR-T in B-cell malignancies | Genetic Engineering - GEG Tech top picks | Scoop.it
A chimeric antigen receptor T-cell therapy induced high response rates across a range of B-cell malignancies, according to phase 1 study results presented at ASH Annual Meeting and Exposition.An interim analysis showed a 93% overall response rate among 15 patients with relapsed or refractory follicular lymphoma who received the investigational therapy.
BigField GEG Tech's insight:

 

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.

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Study may lead to a paradigm shift in the care for adults who relapse from large B-cell lymphoma

Study may lead to a paradigm shift in the care for adults who relapse from large B-cell lymphoma | Genetic Engineering - GEG Tech top picks | Scoop.it
A study published this month in the New England Journal of Medicine (NEJM) may change the standard of care for adults who relapse from large B-cell lymphoma.
BigField GEG Tech's insight:

The CAR T cell-based therapy Yescarta (axicabtagene ciloleucel) and manufactured by Kite Pharmaceuticals, was first approved by the U.S. Food and Drug Administration in 2017 as a third-line treatment for adults with large B-cell lymphoma (LBCL), i.e., for those who had already undergone two cycles of failed treatment. The ZUMA-7 clinical trial was therefore set up to determine whether a single infusion of Yescarta is superior to the existing and long-standing standard of second-line care, which is a stem cell transplant after high-dose chemotherapy to kill the lymphoma. According to the results of a clinical trial published December 11th and presented the same day at the American Society of Hematology annual meeting, this treatment is significantly more effective than the current standard of care in treating people with large B-cell lymphoma who relapse after first-line treatment. About 40% of people with LBCL need such second-line treatment, either because their cancer recurs or does not respond adequately to first-line treatment.

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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.
BigField GEG Tech's insight:

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
Scoop.it!

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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