Vectorology - GEG Tech top picks

The ANCHOR system invented by NeoVirtech has been combined by GEG Tech to tag HIV-1 vectors (Lenti-ONE system) and generate autofluorescent lentivirus genomes able to be tracked in real time in living cells (1).

Following this proof of concept, the collaboration with Francesca Di Nunzio lab at the Pasteur Institute, has resulted a new breakthrough published in Journal of Virology (2): “We are very excited to be able to visualize the pre-integration complex and the fate of the HIV1 genome in living cells. The ANCHORTM technology is for us the state of the art technology allowing for the first time thorough investigation of HIV1 biology and response to antiviral treatment directly in living cells. The technology is the only one compatible with CLEM (correlative light-electron microscopy) to dissect the ultrastructural structure of the HIV1 particle. The fact that the ANCHORTM technology does not modify HIV1 virus biology offers an unprecedented view of the dynamics of this virus in living cells”.

Francesca Di Nunzio Ph.D, Pasteur Institute, Paris, France.

(1) https://www.linkedin.com/pulse/first-ever-live-tracking-hiv-1-(2) based-lentiviral-human-grandchamp/.

https://jvi.asm.org/content/early/2020/04/16/JVI.00135-20

Here the scientists optimised insulin production using lentiviral transduced canine mesenchymal stromal cells (MSCs) aiming to evaluate their ability for use as surrogate beta cells. Lentivirus vectors encoding the proinsulin gene under the control of SFFV, CMV, EF1α and SV40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. Their results suggest that optimised lentiviral transduction of the insulin gene into primary canine MSCs renders these cells capable of secreting insulin both short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.

Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. Here the scientists describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. They show that the percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. They conclude that integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.

GEG Tech provides a large range of IDLVs and also several lines of this type of vector with different intergase mutations (including D64V). 

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This review presents the challenges of up-scaling lentivirus production and processing approaches, novel systems for overcoming these issues, and the quality assessments recommended for producing a clinical grade lentiviral gene therapy product.

In this work, the scientists show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Their results suggest that the reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.