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Kinetics of lentiviral vector transduction in human CD34+ cells

Kinetics of lentiviral vector transduction in human CD34+ cells | Vectorology - GEG Tech top picks | Scoop.it
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To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, the authors evaluated the kinetics of lentiviral transduction in human CD34+ cells. Their data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34+ cells. These findings provide an avenue for optimizing human CD34+ cell transduction.


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Novel method for gene editing of hPSCs yields up to 84% knockout efficiency

Novel method for gene editing of hPSCs yields up to 84% knockout efficiency | Vectorology - GEG Tech top picks | Scoop.it
A Penn State-led team of interdisciplinary researchers has developed techniques to improve the efficiency of CRISPR-Cas9, the genome editing technique that earned the Nobel Prize in 2020.
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Researchers have developed a more efficient and accessible process for applying CRISPR-Cas9 systems in human pluripotent stem cells (hPSCs) that could advance diagnoses and treatments for genetic disorders. To improve this system, the researchers changed the way the tool is delivered to stem cells, using modified RNA (modRNA). ModRNA differs from plasmid DNA in that it replaces one of the basic substrates found in RNA with a chemically modified version, and it is stabilized by a stronger structural support. About 90% of the cells received the modRNA from a single transfection. The researchers also found that the length of time the modRNA was in place was ideal: long enough to modify the cells, but not too long to cause off-target activity. However, when the Cas9 modRNA is successfully delivered to the target gene, it creates a double-stranded break in the genome, which some cells will try to repair and pass the repair, or "mutation," to their offspring. To reduce the toxic side effects of Cas9 and help the modified cells survive, the researchers also introduced a small protein known to help cells grow. This added protein inhibited cell death and improved the efficiency of Cas9 editing by up to 84%. 

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New dual-plasmid editing system for DNA-based information rewriting in vivo

New dual-plasmid editing system for DNA-based information rewriting in vivo | Vectorology - GEG Tech top picks | Scoop.it
DNA-based information is a new interdisciplinary field linking information technology and biotechnology.
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Despite DNA's promise of high stability, high storage density and low maintenance cost, researchers face problems in accurately rewriting the digital information encoded in DNA sequences. To solve the rewriting problem, the researchers established an in vivo double plasmid system using a rationally designed coding algorithm and an information editing tool. This dual-plasmid system is suitable for storing, reading, and rewriting various types of information, including text, codebooks and images. It fully explores the encoding capability of DNA sequences without the need for addressing indices or backup sequences. It is also compatible with various types of coding algorithms, allowing high coding efficiency. For example, the coding efficiency of the current system reaches 4.0 bits per nucleotide. To achieve high efficiency and reliability in rewriting complex information stored in exogenous DNA sequences in vivo, a variety of CRISPR-associated proteins (Cas) and recombinases were used. The information rewriting tool thus became highly adaptable to complex information, resulting in a rewriting reliability of up to 94%, which is comparable to existing gene editing systems. 

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New DNA repair vehicle used to fix a common genetic cause of hereditary kidney disease

New DNA repair vehicle used to fix a common genetic cause of hereditary kidney disease | Vectorology - GEG Tech top picks | Scoop.it
Genetic mutations which cause a debilitating hereditary kidney disease affecting children and young adults have been fixed in patient-derived kidney cells using a potentially game-changing DNA repair-kit. The advance developed by University of Bristol scientists is published in Nucleic Acids Research.
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Podocin is a protein normally located on the surface of specialized kidney cells and is essential for kidney function. The defective podocin, however, gets stuck inside the cell and never reaches the surface, permanently damaging the podocytes, a common genetic cause of inherited steroid-resistant nephrotic syndrome (SRNS). Gene therapy that repairs the genetic mutations that cause defective podocin offers hope to patients. However, current delivery systems that include lentiviruses, adenoviruses and adeno-associated viruses all share the same limitation in that they are limited in the space inside their viral shells and this in turn limits the amount of cargo they can deliver.  Applying synthetic biology techniques, the researchers therefore redesigned the baculovirus, a human-friendly insect virus that is no longer limited by cargo capacity. By creating a DNA repair kit, including the CRISPR/Cas 9 system and DNA sequences to replace the defective gene. The team delivered a single modified baculovirus with a healthy copy of the podocin gene together with the CRISPR/Cas machines to insert it with base-pair precision into the genome and this was able to reverse the pathogenic phenotype and restore podocin to the cell surface.

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Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy

Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy | Vectorology - GEG Tech top picks | Scoop.it
We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gen
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Many drugs cannot cross the blood-brain barrier (BBB), which also applies to most CRISPR-carrying vehicles. So Bingyang Shi made chemically functional nanocapsules to penetrate the BBB, according to a study recently published in Science Advances. He essentially packaged unique CRISPR-Cas9/sgRNA complexes in a coating consisting of a thin polymeric shell made of positively charged guanidine acrylate cross-linked with N,N-bis(acryloyl)cystamine that contains biodegradable disulfide bonds. The shell was finally decorated with the peptide angiopep-2. While coating rather than encapsulation was chosen to maintain the particle size at approximately 30 nm and thus facilitate BBB penetration, the angiopep-2 peptide served two purposes. It is a ligand that binds to low-density lipoprotein receptor-related protein-1, which is most highly expressed on BBB endothelial cells and glioblastoma (GBM) cells. This will result in the concentration of the nanocapsules around the BBB, increasing their chance of penetration into the brain, where they will then be preferentially taken up by GBMs. However, more work is needed before these nanocapsules can emerge as a therapy for GBM patients.

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Estrogen receptor positive breast cancers have patient specific hormone sensitivities and rely on progesterone receptor | Nature Communications

Estrogen receptor positive breast cancers have patient specific hormone sensitivities and rely on progesterone receptor | Nature Communications | Vectorology - GEG Tech top picks | Scoop.it
Estrogen and progesterone receptor (ER, PR) signaling control breast development and impinge on breast carcinogenesis. ER is an established driver of ER + disease but the role of the PR, itself an ER target gene, is debated. We assess the issue in clinically relevant settings by a genetic approach and inject ER + breast cancer cell lines and patient-derived tumor cells to the milk ducts of immunocompromised mice. Such ER + xenografts were exposed to physiologically relevant levels of 17-β-estradiol (E2) and progesterone (P4). We find that independently both premenopausal E2 and P4 levels increase tumor growth and combined treatment enhances metastatic spread. The proliferative responses are patient-specific with MYC and androgen receptor (AR) signatures determining P4 response. PR is required for tumor growth in patient samples and sufficient to drive tumor growth and metastasis in ER signaling ablated tumor cells. Our findings suggest that endocrine therapy may need to be personalized, and that abrogating PR expression can be a therapeutic option. The role of progesterone receptor (PR) and its interplay with estrogen receptor (ER) in breast cancer is controversial. Here, the authors demonstrate that PR can have an ER-independent role in breast cancer growth and metastasis and that its effects are dependent on MYC and androgen receptor signatures.
BigField GEG Tech's insight:

Proud to share one of our long-time partner's publication whom we have designed robust and efficient lentiviral vectors for its project in breast cancer.

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Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates | Nature Communications

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates | Nature Communications | Vectorology - GEG Tech top picks | Scoop.it
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In this study the scientists developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. 

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CRISPR/Cas9 deletions induce adverse on-target genomic effects

CRISPR/Cas9 deletions induce adverse on-target genomic effects | Vectorology - GEG Tech top picks | Scoop.it
Researchers utilized a CRISPR/Cas9 system to evaluate the usage of tRNA by deleting two tRNA genes from the genomes of hyper hepatocellular carcinoma and human near-haploid chronic myeloid leukemia cells.
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In a recent study, a team of researchers used a CRISPR/Cas9 system to evaluate the use of tRNA by deleting two tRNA genes from the genomes of hyperhepatocellular carcinoma and human chronic myeloid quasi-haploid leukaemia cells. The authors discovered numerous unexpected genomic changes at the target region using an improved droplet-based target enrichment approach followed by Oxford Nanopore Technology long-read sequencing. The method used in this study demonstrates that CRISPR/Cas9 can lead to the integration of endogenous and exogenous DNA fragments and also produce local inversions, duplications and insertions of functional target-derived fragments. This research presents evidence that a combination of duplication and inversion, as well as integration of exogenous DNA fragments and clustered interchromosomal rearrangements, can occur simultaneously. Furthermore, it was shown for the first time that the target-derived fragments were nevertheless functional despite these modifications, which may complicate mechanistic explanations. These results reveal a new example of unintended CRISPR/Cas9 editing events that can go unnoticed and have a significant impact on the conclusions drawn from experimental reads. 

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Clinical Trial Update: CRISPR Therapy Designed to Cure Sickle Cell Disease

Clinical Trial Update: CRISPR Therapy Designed to Cure Sickle Cell Disease | Vectorology - GEG Tech top picks | Scoop.it
This clinical update looks at Graphite Bio's sickle cell disease candidate GPH101. GPH101 is an ex vivo CRISPR-edited cell therapy that is anticipated to provide a permanent cure by targeting the root cause of disease. Clinical trial enrolment is ongoing at multiple sites.
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GPH101 is an ex vivo CRISPR-edited cell therapy designed to correct the HBB point mutation and should provide a permanent cure by targeting the root cause of the disease.  Correction of the HBB gene results in a decrease in sickle cell hemoglobin production and restores normal expression of adult hemoglobin, thereby restoring normally functioning red blood cells. The candidate is engineered using patient-derived hematopoietic stem cells (HSCs) that are edited with Cas9-sgRNA gene editing machines and a DNA repair model, in a mechanism that essentially cuts the mutation from the genome and replaces it with the correct sequence. Cas9-sgRNA is delivered by ribonucleoprotein (RNP) complexes while delivery of a corrected DNA template is achieved by an adeno-associated virus 6 (AAV6) vector. The FDA approved an IND application in December 2020 for GPH101 and it will be evaluated in the Phase 1/2 CEDAR trial, an open-label, multicenter study to evaluate the safety, efficacy, and pharmacodynamics of the new therapeutic candidate in adults and adolescents with severe sickle cell disease. The trial will enroll approximately 15 adult and adolescent participants at up to five clinical trial sites in the United States.

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Using mRNA to introduce gene editor into human muscle stem cells

Using mRNA to introduce gene editor into human muscle stem cells | Vectorology - GEG Tech top picks | Scoop.it
Mutations that lead to muscle atrophy can be repaired with the gene editor CRISPR-Cas9. A team led by ECRC researcher Helena Escobar has now introduced the tool into human muscle stem cells for the first time using mRNA, thus discovering a method suitable for therapeutic applications.
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Muscular dystrophies are currently incurable. However, the mutations that lead to muscle atrophy could be repaired with the CRISPR-Cas9 gene editor. A team led by the ECRC researcher introduced the tool into human muscle stem cells for the first time using mRNA, discovering a method suitable for therapeutic applications. First, to introduce the mRNA into the stem cells, the researchers used an electroporation process, which temporarily makes cell membranes more permeable to larger molecules. In the next step, the team used a deliberately modified molecule on the surface of human muscle stem cells to show that the method can be used to correct genetic defects in a targeted manner. Finally, the team tested a tool similar to the CRISPR-Cas9 gene editor that does not cut DNA, but only modifies it in one place with extreme precision. In particular, the researchers showed that the corrected muscle stem cells are just as capable as healthy cells of fusing together and forming young muscle fibers.  

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Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics | Nature Neuroscience

Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics | Nature Neuroscience | Vectorology - GEG Tech top picks | Scoop.it

The mammalian brain contains many specialized cells that develop from a thin sheet of neuroepithelial progenitor cells. Single-cell transcriptomics revealed hundreds of molecularly diverse cell types in the nervous system, but the lineage relationships between mature cell types and progenitor cells are not well understood. Here we show in vivo barcoding of early progenitors to simultaneously profile cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. By reconstructing thousands of clones, we discovered fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. We combined spatial transcriptomics with clonal barcoding and disentangled migration patterns of clonally related cells in densely labeled tissue sections. Our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture. Ratz et al. present an easy-to-use method to barcode progenitor cells, enabling profiling of cell phenotypes and clonal relations using single-cell and spatial transcriptomics, providing an integrated approach for understanding brain architecture.

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Proud to share one of our long-time partner publication whom we have designed very specific custom lentiviral vectors.

@Micheal Ratz
@Jonas Frisén
@Joakim Lundeberg
@Leonie von Berlin
@Gioele La Manno
@Jakub Orzechowski Westholm
@Ludvig Larsson

The mammalian brain contains many specialized cells that develop from a thin sheet of neuroepithelial progenitor cells. However, the lineage relationships between mature cell types and progenitor cells are not well understood. Therefore, researchers performed high-throughput clonal tracking and expression profiling of mouse forebrain cells using single-cell and spatial transcriptomics and found two populations of fate-restricted progenitor cells present as early as embryonic day (E) 9.5 in the murine hippocampus. They found that microglia are generated from a limited number of progenitor cells that undergo massive clonal expansion as well as widespread migration throughout the mouse telencephalon. The results thus demonstrate the utility of high-throughput clonal tracing in the mouse brain to provide molecular information about brain development at the single cell and tissue level. TREX is the technique that allowed them to profile TRacking and gene EXpression of clonally bound cells in the mouse brain by high-throughput single-cell RNA sequencing (scRNA-seq). TREX is based on a diverse library of lentiviruses containing barcodes downstream of a GFP protein.

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Nonviral CAR-T is ‘remarkably safe’ and clinically active in B-cell ALL

Nonviral CAR-T is ‘remarkably safe’ and clinically active in B-cell ALL | Vectorology - GEG Tech top picks | Scoop.it
More than 60% of patients with relapsed or refractory B-cell acute lymphoblastic leukemia achieved initial complete remission after receiving an investigational chimeric antigen receptor T-cell therapy, phase 1/phase 2 study results showed.By their initial 1-month evaluation, 73% of patients who received the highest dose levels had a complete response to therapy, according to findings presented
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CARCIK-CD19, an investigational therapy developed by scientists in Italy, involves donor-derived CD19-targeted CAR T cells differentiated according to the cytokine-induced killer (CIK) cell protocol that uses non-viral Sleeping Beauty transposon gene transfer to prevent graft-versus-host disease. More than 60% of patients with relapsed or refractory B-cell acute lymphoblastic leukemia achieved initial complete remission after receiving experimental chimeric antigen receptor T-cell therapy, results from the phase 1/phase 2 study showed. At their initial one-month assessment, 73% of patients who received the highest doses had a complete response to treatment, according to results presented at the 4th European Society for Blood and Marrow Transplantation-European Hematology Association CAR-T Cell Meeting.  

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Discovery and validation of human genomic safe harbor sites for gene and cell therapies - Cell Report

Discovery and validation of human genomic safe harbor sites for gene and cell therapies - Cell Report | Vectorology - GEG Tech top picks | Scoop.it
Existing approaches to therapeutic gene transfer are marred by the transient nature of gene expression following non-integrative gene delivery and by …
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The efficacy, persistence and predictability of many future gene and cell therapies to treat diseases such as cancer, rare genetic diseases could be enhanced by what are called "genomic safe harbors" (GSHs). These are landing sites in the human genome that can safely accommodate new therapeutic genes without causing other unintended changes in a cell's genome that could pose a risk to patients. To date, only a few candidate GSHs have been explored, and they all come with some caveats. However, a collaboration of researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard, Harvard Medical School (HMS) and ETH Zurich in Switzerland, has developed a computational approach to identify GSH sites with significantly higher potential for safe insertion of therapeutic genes and their sustained expression in multiple cell types. For two of the 2,000 predicted GSH sites, the team provided extensive validation with adoptive T cell therapies and in vivo gene therapies for skin diseases in mind. By modifying the identified GSH sites to carry a reporter gene in T cells and a therapeutic gene in skin cells, respectively, using CRISPR-Cas9, they demonstrated safe and sustained expression of the newly introduced genes. The study is published in Methods of Cell Reporting. 

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News: CRISPR-Cas13d Nuclease System Effectively Inhibits HIV-1 Replication In Vitro

News: CRISPR-Cas13d Nuclease System Effectively Inhibits HIV-1 Replication In Vitro | Vectorology - GEG Tech top picks | Scoop.it
Researchers at Texas Biomedical Research Institute in San Antonio, Texas have developed a novel strategy to inhibit human immunodeficiency virus (HIV) replication, using guide RNAs that target highly conserved regions of HIV-1 in conjunction with the CRISPR-Cas13d nuclease system.
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A recent publication in Viruses detailed a CRISPR-based strategy to effectively target and control HIV-1 infections in vitro with CasRx. Dr. Smita Kulkarni's laboratory at the Texas Biomedical Research Institute successfully combined a novel set of guide RNAs (gRNAs), known as polyHIV, with CasRx to target and inhibit HIV-1 replication. This work demonstrates for the first time the potential of the CRISPR-Cas13d nuclease system to target acute and latent HIV infections that could potentially be used to improve the effectiveness of current HIV treatment options. Kulkarni's team strategically selected four sites in the HIV genome that are more than 70% conserved from the HIV-1 transcript sequences deposited in the LANL database and specific gRNAs were designed against these sites. The four gRNAs were selected from 20 candidates to form a poly-gRNA assembly (polyHIV) and paired with CasRx, a Cas protein derived from the Cas13d family with high RNA cleavage activity. The selected target sites were non-overlapping and located in the gag, pol, protease (prot), integrase (int), cPPT and central termination sequence (CTS) coding regions in the HIV-1 genome. The study is the first to provide a single vector with four gRNAs targeting non-overlapping conserved regions of HIV-1.

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News: CRISPR Gene Silencing Offers Hope for Ultra-Rare Muscular Dystrophies

News: CRISPR Gene Silencing Offers Hope for Ultra-Rare Muscular Dystrophies | Vectorology - GEG Tech top picks | Scoop.it
Patients with collagen VI-related disorders experience progressive and debilitating disease. New research findings from Spain’s Institut de Recerca Sant Joan de Déu (IRSJD) provides new hope for these diseases, demonstrating the power of CRISPR-Cas9 to silence a dominant negative mutation in patient fibroblasts.
BigField GEG Tech's insight:

Collagen VI-related dystrophies (COL6-RD) are caused by mutations in the COL6A1, COL6A2 and COL6A3 genes that encode the alpha chains of collagen VI, a key component of the extracellular matrix. The prevalent pathogenic mutation in COL6A1 is a single nucleotide substitution, where a glycine is replaced by an arginine in the N-terminus of the triple helix domain which affects the folding of the resulting protein, hindering the association of tetramers to create the necessary collagen VI microfibrils. Moreover, there is currently no effective treatment available for COL6-RD. However, a recent paper demonstrates the use of CRISPR-Cas9 gene editing to mitigate the pathogenic effects of a dominant negative mutation in the COL6A1 gene. Nevertheless, an obvious hurdle for this type of therapy is the delivery of CRISPR-Cas9 reagents to fibroblasts in vivo; successful editing of somatic tissues such as muscle remains a significant challenge in the broader field. The research team hopes that recent advances in several areas of CRISPR delivery vehicles, including muscle adeno-associated trophic virus vectors and tissue-specific nanoparticles, may be the answer.

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Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport 

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport  | Vectorology - GEG Tech top picks | Scoop.it
The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed t
BigField GEG Tech's insight:

Proud to share a publication of one of our partner. In this study, they used a very specific vector allowing the expression of the Alternative OXidase (AOX) from Ciona intestinalis.

This type of vector played a part about the in vitro and in vivo characterization of the melatonin effects on the reactive oxygen species (ROS) levels in cancer cells .

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Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence | Nature Communications

Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence | Nature Communications | Vectorology - GEG Tech top picks | Scoop.it
More than 70% of human breast cancers (BCs) are estrogen receptor α-positive (ER+). A clinical challenge of ER+ BC is that they can recur decades after initial treatments. Mechanisms governing latent disease remain elusive due to lack of adequate in vivo models. We compare intraductal xenografts of ER+ and triple-negative (TN) BC cells and demonstrate that disseminated TNBC cells proliferate similarly as TNBC cells at the primary site whereas disseminated ER+ BC cells proliferate slower, they decrease CDH1 and increase ZEB1,2 expressions, and exhibit characteristics of epithelial-mesenchymal plasticity (EMP) and dormancy. Forced E-cadherin expression overcomes ER+ BC dormancy. Cytokine signalings are enriched in more active versus inactive disseminated tumour cells, suggesting microenvironmental triggers for awakening. We conclude that intraductal xenografts model ER + BC dormancy and reveal that EMP is essential for the generation of a dormant cell state and that targeting exit from EMP has therapeutic potential. The study of tumour dormancy is limited by suitable in vivo models. Here the authors show that mammary intraductal breast cancer (BC) xenografts model estrogen receptor α-positive (ER+) BC dormancy and rapid metastatic progression characteristic of triple-negative (TN) BC. The dormant disseminated ER+ BC cells display characteristics of epithelial-mesenchymal plasticity and forced expression of E-cadherin allows them to overcome dormancy.
BigField GEG Tech's insight:

Once again, proud to share a high-quality publication of the Brisken Lab at the EPFL, one of our long-standing partners. Two publications in Nature in two months, Wow ! As you said Cathrin, “tons more to do together” !

I take the opportunity to introduce the next workshop organized by Cathrin Brisken about the Preclinical and Personalized Breast Cancer Research.

 

https://www.epfl.ch/labs/brisken-lab/preclinicalmodelcourse/

 

First publication: https://www.linkedin.com/posts/nicolas-grandchamp-ab169a19_estrogen-receptor-positive-breast-cancers-activity-6943209061161017345-7_5P?utm_source=share&utm_medium=member_desktop

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Evaluating the State of the Science for Adeno-Associated Virus (AAV) Integration: An Integrated Perspective - CellPress

Evaluating the State of the Science for Adeno-Associated Virus (AAV) Integration: An Integrated Perspective - CellPress | Vectorology - GEG Tech top picks | Scoop.it
On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, fea…
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The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance and bioethics are discussed.

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Gene therapy’s comeback: how scientists are trying to make it safer - Nature

Gene therapy’s comeback: how scientists are trying to make it safer - Nature | Vectorology - GEG Tech top picks | Scoop.it
Unwanted immune responses threaten to derail some gene therapies. But researchers are seeking ways to combat harmful inflammation.
BigField GEG Tech's insight:

It is a pity that non integrating lentiviral vectors are unknown or underused. This is not THE solution but in several cases, I think their use would be more relevant than AAVs use in term of immune response. The advancements of gene therapy will go through the design and development of complementary vector platforms.

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New method increases efficiency of gene editing while minimizing DNA deletion sizes

New method increases efficiency of gene editing while minimizing DNA deletion sizes | Vectorology - GEG Tech top picks | Scoop.it
Wake Forest Institute for Regenerative Medicine (WFIRM) scientists working on CRISPR/Cas9-mediated gene editing technology have developed a method to increase efficiency of editing while minimizing DNA deletion sizes, a key step toward developing gene editing therapies to treat genetic diseases.
BigField GEG Tech's insight:

Although CRISPR/Cas9 mainly generates short insertions or deletions at the target site, it can also make large deletions of DNA around the specific target site. These large deletions pose safety concerns and can reduce the efficiency of functional editing. The WFIRM team is looking at ways to reduce the risk of this happening. The research described in their recent paper, published recently in Nucleic Acids Research, aimed to combat the generation of unpredictable long DNA deletions on target and find a way to prevent this, a key step towards the development of gene editing therapies to treat genetic diseases. The team evaluated a variety of human cells and genes of interest and found that fusing DNA polymerase I or the Klenow fragment to the Cas9 enzyme minimised large, unanticipated deletions of genomic DNA without sacrificing the efficiency of genome editing. On the contrary, it even increased editing efficiency in primary human cells.  

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 Nasal Spray Sends CRISPR to the Brain

 Nasal Spray Sends CRISPR to the Brain | Vectorology - GEG Tech top picks | Scoop.it
Most pharmaceuticals don't reach the brain, leaving cognitive impairments due to diseases like mucopolysaccharidoses (MPS) hard to treat. Now, a Brazilian research team has developed a method for nasal delivery of CRISPR reagents to the brain that improves MPS cognitive symptoms in mice. Experiments will proceed with monkeys later this year.
BigField GEG Tech's insight:

Mucopolysaccharidosis I is caused by mutations in the gene for the enzyme alpha-L-iduronidase (IDUA). This enzyme is required for the breakdown of substances called glycosaminoglycans (GAGs) that are by-products of chemical reactions in the body's cells. In 2018, a team of researchers was able to demonstrate the feasibility of CRISPR gene editing in vivo in mice via intravenous injection. The treatment partially restored IDUA enzyme activity in organs such as the heart, lungs, liver and kidneys, but not in the brain because the blood-brain barrier protects it. However, this is a problem for patients with the severe form of the disease who may develop cognitive decline. The researchers therefore had the idea of going through the nose to reach the olfactory bulb just above.  As a result of the experiments, it was finally shown that non-invasive administration of CRISPR reagents via nasal administration allows gene editing and partial restoration of IDUA activity in the brain and improved cognitive function, while biomarkers of the disease in other organs also improved.

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Rapid manufacturing of non-activated potent CAR T cells | Nature Biomedical Engineering

Rapid manufacturing of non-activated potent CAR T cells | Nature Biomedical Engineering | Vectorology - GEG Tech top picks | Scoop.it
Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability. Potent chimaeric antigen receptor T cells can be generated within one day from T cells derived from peripheral blood without the need for T-cell activation.
BigField GEG Tech's insight:

In the process of engineering CAR T cells, if the patient's T cells are out of the body for too long, they can lose their ability to replicate, which is critical to their effectiveness as a live drug. Thus, a research team has developed a new approach that reduces the time needed to modify patients' immune cells so that they can be quickly reinjected into the body. The manufacturing process for CAR T cells typically takes between nine and 14 days, whereas this new process would reduce the manufacturing time to as little as 24 hours. Traditional manufacturing approaches require that T cells be stimulated in a way that encourages the cells to replicate and grow in number. The key to the Penn researchers' manufacturing approach is the lentiviral vector that delivers the CAR gene to T cells. Lentiviral vectors are able to transfer genes like the CAR gene to cells without needing this initial "activation" step. This approach has the dual advantage of speeding up the overall manufacturing process while maintaining T-cell potency. The team hopes that the reduced manufacturing time could make the therapy more cost-effective and accessible to more patients.

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News: First Patient Dosed in CRISPR Trial for Acute Myleoid Leukaemia

News: First Patient Dosed in CRISPR Trial for Acute Myleoid Leukaemia | Vectorology - GEG Tech top picks | Scoop.it
Intellia Therapeutics recently announced that the first patient had been dosed in its Phase 1/2a CRISPR trial for acute myleoid leukaemia (AML). The new therapeutic candidate, NTLA-5001, is a CRISPR-edited T cell receptor therapy designed to targeted Wilm's Tumour (WT1) antigen, which is found on AML and several other blood cancers as wel
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Intellia Therapeutics recently announced that the first patient has received a dose in its Phase 1/2a CRISPR trial for acute myeloid leukemia (AML). The new therapeutic candidate, NTLA-5001, is a CRISPR-edited T-cell receptor therapy designed to target Wilm's tumor antigen (WT1), which is found on AML and several other blood cancers as well as certain types of solid cancer. To generate NTLA-5001, T cells are isolated from a patient with AML and the T cell receptor (TCR) is replaced with a naturally occurring high avidity TCR (i.e. from a healthy donor) with specificity for WT1. Complete elimination of the endogenous TCR is achieved by CRISPR-Cas9-mediated replacement of the TRAC locus with the WT1 TCR, and subsequent site-specific inactivation of the TRBC locus, which encodes the T cell receptor beta chains. For TCR locus disruption, SpCas9 and gRNAs are delivered to cells via lipid nanoparticles. The WT1 TCR is delivered to cells via adeno-associated virus (AAV) transduction. NTLA-5001 is therefore being evaluated in a multi-center, single-dose Phase 1/2a study that will assess its safety, tolerability, cellular kinetics (CK), activity and pharmacodynamics (PD) in AML participants. 

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Disrupting off-target Cas9 activity in the liver | Nature Biomedical Engineering

Disrupting off-target Cas9 activity in the liver | Nature Biomedical Engineering | Vectorology - GEG Tech top picks | Scoop.it
Off-target genome editing in the liver can be reduced by using lipid nanoparticles to deliver oligonucleotides that disrupt the secondary structure of single-guide RNAs as well as short interfering RNAs targeting Cas9 mRNA.
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In genome editing, it is crucial to ensure target-specific editing in diseased cells and to avoid altering the genome of spectator cells. Since a substantial fraction, i.e. 30-90%, of an intravenously injected dose of lipid nanoparticles (LPNs) accumulates in the liver, off-target editing in this organ must be minimized. According to a study published in Nature Biomedical Engineering , LNPs can be used to deliver two types of anti-CRISPR nucleic acid to the liver to disrupt genome editing in hepatocytes, thereby improving the specificity of CRISPR-Cas genome editing in the extrahepatic tissues and organs.  To determine whether anti-CRISPR oligonucleotides could be used to reduce off-target editing in the liver, researchers first treated mice stably expressing Cas9 tagged with GFP with anti-CRISPR or scrambled oligonucleotides formulated in NLPs that preferentially target the liver. Two hours later, they treated the same mice with the same NPLs, but instead encapsulated gRNA targeting the GFP locus. Compared to scrambled oligonucleotides, the anti-CRISPR ones reduced the frequency of deletion mutations in hepatocytes by more than twofold. Therefore, Cas9-mediated on-target genome editing in extrahepatic tissues could be maintained while limiting off-target editing in the liver. 

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Novel approaches to malignant brain tumor treatment aim to overcome familiar challenges

Novel approaches to malignant brain tumor treatment aim to overcome familiar challenges | Vectorology - GEG Tech top picks | Scoop.it
Hemonc Today | Although therapeutic advances during the past decade have dramatically improved cancer outcomes overall, the outlook for patients with malignant brain tumors has remained poor.The 5-year relative survival rate for these patients increased only from 23% between 1975 and 1977 to 36% between 2009 and 2015, with rates in the single digits for those with particularly lethal brain tumor types,
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The challenge in treating brain tumors has been the inability of drugs to penetrate the blood-brain barrier. In addition, the use of treatments that can cross the barrier, such as chemotherapy and radiation therapy, is often limited due to their toxicity. Other standard treatments, such as surgery, are not even an option for some patients because of the location of the tumor. Among brain tumors, low-grade pediatric ones have specific mutations that can be effectively targeted with a single-pathway drug. However, this is not the case for high-grade pediatric brain tumors. Therefore, Vitanza and colleagues are conducting three trials at Seattle Children's, including pediatric patients with predominantly lethal disease that has progressed after initial standard treatment. BrainChild-01 is examining the use of locoregionally administered HER2-directed CAR T cells; BrainChild-02 is using EGFR-specific CAR T cells to treat children and young adults with relapsed or refractory EGFR-positive central nervous system tumors; and BrainChild-03 is using B7-H3-targeted CAR T cells for all patients with recurrent CNS tumors and those with the deadly brain tumor diffuse intrinsic glioma. 

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Decade-long leukaemia remissions with persistence of CD4+ CAR T cells - Nature

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells - Nature | Vectorology - GEG Tech top picks | Scoop.it
The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1–7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1–4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia. Infusion of CD19-directed chimeric antigen receptor T cells into two patients with chronic lymphocytic leukaemia resulted in complete tumour remission and persistence of the infused cells more than ten years later.
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Good News ?

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