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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
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.
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.
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.
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.
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.
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
Existing approaches to therapeutic gene transfer are marred by the transient nature of gene expression following non-integrative gene delivery and by …
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.
The ability to induce genome editing of the vascular endothelium has been challenging.
Zhang et al. show that PP/PEI nanoparticles could deliver plasmid DNA to adult mice
for robust genome editing and 80% knockdown of protein expression selectively in ECs
of various vascular beds and also for transgene expression simultaneously.
Sonodynamic therapy uses ultrasound in combination with drugs to release harmful reactive oxygen species (ROS) at the site of a tumor.
Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR–Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy. Nanoparticle-mediated photoporation is used to temporarily permeabilize cell membranes for intracellular delivery of macromolecules, but cell exposure to nanoparticles might cause cellular damage and hamper application of the technique to therapeutic cell engineering. Here the authors show that, under photothermal heating, nanofibre-embedded iron oxide nanoparticles can be used to deliver effector macromolecules to different types of cells, in a contactless manner, with no cellular toxicity or diminished therapeutic potency.
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On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, fea…
Unwanted immune responses threaten to derail some gene therapies. But researchers are seeking ways to combat harmful inflammation.
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.
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.
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.
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
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.
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,
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.
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes. Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.
Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes. Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.
This up-to-date roundup presents the 7 clinical-stage gene-editing approaches to sickle cell disease, which affects millions of people worldwide and is the most common inherited blood disorder in the United States. Current clinical-stage approaches to treating this disease include CRISPR-Cas9, CRISPR-Cas12a, and base editing.
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Scoop.it!
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.