Vectorology - GEG Tech top picks

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. 

Four early-phase studies being presented today during the 61st annual meeting of the American Society of Hematology (ASH) highlights the rapid advances being made in cellular immunotherapy for the treatment of hematological malignancies.

Here, the scientists report the development of an alternative safeguard for chimeric antigen receptor T-cell therapies. They have incorporated a safeguard component into a conventional CAR architecture that enables tight control over CAR T-cell activity and has the potential to improve their safety profile in clinical settings. In addition, the CubiCAR architecture is compatible with multiple scFvs designed against different targets, thus bearing the potential to improve the safety of CAR T-cell immunotherapies for a broad range of patients.

The safety of lentiviral vectors is a factor in their acceptance as clinical therapies. In this issue of Molecular Therapy, Cornetta et al. (2017) screened 460 cell products for replication-competent lentivirus (RCL); none were positive. The low risk of RCL suggests that revisions to U.S. FDA testing guidelines are warranted.

Precise determination of tumour growth is a prerequisite for estimating the functional importance of tumour suppressor alterations in cancer. A study in Nature Methods now describes the development of Tuba-seq, an approach that integrates tumour barcoding with high-throughput sequencing, to precisely quantify the size of individual tumours in vivo.

The new method relies on the use of lentiviral Cre-vector libraries that contain DNA barcodes to stably tag individual lesions with a unique identifier. In a genetically engineered mouse model of human lung cancer, transduction of lung epithelial cells leads to Cre recombinase-mediated expression of oncogenic Kras. Expression of this oncogene induces the simultaneous development of multiple tumours, each now tagged with a unique 15-nucleotide (nt) barcode. In a first set of experiments, the Kras-driven tumours were analysed side-by-side with lung tumours that carried additional deficiencies in the tumour suppressor genes Lkb1 or p53. High-throughput sequencing was performed on bulk tumour DNA, and read counts from barcodes were converted to estimated cell numbers based on mixed-in 'benchmark' cells. Tuba-seq accurately determined the number of neoplastic cells in all tumours in parallel, finding them to be more than 1,000-fold different in cell number. The size distribution of tumours further provided insight into the different mechanisms of action of p53 and LKB1 (also known as STK11) in suppressing tumour growth.

Having demonstrated the utility of Tuba-seq, Rogers, McFarland, Winters et al. set out to adapt the method for multiplexing, this time using CRISPR–Cas9-mediated inactivation of 11 known or suspected tumour suppressors in the Kras-driven lung adenocarcinoma model. Lentiviral vectors carried an 8-nt tag specific for an encoded guide RNA, as well as the 15-nt barcode for cancer cell quantification. In this experimental setup, in which different tumour suppressor genotypes were present within the same mouse, size and genotype of tumours were simultaneously determined by sequencing the guide RNA-specific barcode tag. Lkb1, Rb1, Cdkn2a and Apc were confirmed as tumour suppressors in Kras-driven lung cancer growth, and novel potential lung tumour suppressors (splicing factor Rbm10 and the methyltransferase Setd2) were found.

With its exceptional level of precision and reproducibility, Tuba-seq will be a powerful tool to determine the impact of loss-of-function mutations on tumour growth in vivo. Moreover, this approach can likely be adapted to other types of cancers and scaled up to even more extensively multiplexed libraries.

In this wotk, the scientists investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. They demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Their results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

In this study, the scientists design a lentiviral vector to overexpress SOD2 under the control of a chimeric promoter C9BC/CMV. In vitro experiments revealed that radiation-induced SOD2 overexpression inhibited tumor cell proliferation and decreased apoptosis among normal cells as compared to untransfected cells. Similar effects were observed in vivo. Thus radiation-induced SOD2 overexpression via the chimeric C9BC promoter increased the radiosensitivity of HT-29 human colorectal cancer cells and concurrently protected normal CCD 841 CoN colorectal cells from radiation damage.

CAR-T has become a hype within the biotech field recently. Companies promising to deliver this new generation of therapies are shining on stock markets, sometimes leading to billion-dollar valuations. But the rise of CAR-Ts would not have been possible without a crucial technology: the lentiviral vector!

In this study, the authors used a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). They used it to facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with. Their results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL.

Here the authors used a lentiviral vector to investigate non-invasive in vivo monitoring of CD44-positive cancer stem-like cells in breast cancer by γ-irradiation. They generated a breast cancer cell line stably expressing fLuc gene by use of recombinant lentiviral vector controlled by CD44 promoter (MCF7-CL). They irradiated MCF7-CL spheres and observed the effects by immunofluorescence and flow cytometry. Their results indicate that increased CD44 expression, caused by general feature of CSCs by irradiation and sphere formation, can be monitored by using bioluminescence imaging. This system could be useful to evaluate CD44- expressed CSCs in breast cancer by BLI in vivo as well as in vitro for radiotherapy.

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.

A clever strategy which could be very useful to design the next generations of cancer immunotherapy, including for solid tumors .

GEG Tech has been one of the first actors to provide solutions for CAR cells generation. By the way, its last generation of RNA delivery system based on lentiviral vectors could highly improve this type of strategy, taking the advantages of the lenti platform without the biosaftey issues.

quick zap of electricity makes T cells more receptive to taking in new genetic material and gene-editing reagents, researchers report July 11 in Nature. The discovery could expedite protocols for creating immunotherapies to treat a range of cancers.

Researchers at Seattle Children’s Hospital have launched a first-in-human trial to investigate whether infusions of T-cell antigen–presenting cells prolong the persistence of chimeric antigen receptor T-cell immunotherapy among children with relapsed or refractory acute lymphoblastic leukemia or lymphoma.

In this article, Okano, Nakamura and colleagues report that the iCaspase9 system abolished
hiPSC-NS/PCs-derived tumors and controlled adverse events. The iCaspase system may
serve as an important countermeasure against post-transplantation adverse events in
stem cell transplant therapies.

This review focuses on CARs recognizing the B cell antigen CD19. Both retro- and lentiviral vectors are used encoding the different anti-CD19 CAR constructs comprising costimulatory molecules like CD28, CD137/4-1BB and OX40 either alone (2nd generation CARs) or in combination (3rd generation CARs). Current up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and Non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 59 ongoing trials is given.

In this work, the authors design lentiviral vectors expressing tyrosinase (TYR) driven by an optimized human telomerase reverse transcriptase (hTERT) promoter or a cytomegalovirus(CMV) promoter in the hopes of performing noninvasive and real-time tumor-specific imaging. Their data show that this optimized hTERT promoter-driving tyrosinase expression system might be a useful diagnostic tool for the detection of tumors using MR imaging.

The present study used a rat model of bone cancer pain in order to investigate whether lentiviral mediated delivery of small interfering RNAs targeting GluN2B (LVGluN2B) could attenuate pain associated with bone cancer, by selectively decreasing GluN2B expression within the rostral anterior cingulate cortex. The results suggest that GluN2B may be a potential target for RNA interference therapy for the treatment of pain associated with bone cancer. Furthermore, the lentiviral vector delivery strategy may be a promising novel approach for the treatment of bone cancer pain.

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