Vectorology - GEG Tech top picks

A new precision-targeted delivery method for CRISPR-Cas9, published in the journal Nature Biotechnology, enables the genetic editing of highly specific subsets of cells while still inside the body: a step towards a programmable delivery method that would eliminate the need to erase patients' bone marrow and immune systems before giving them modified blood cells. The delivery method, developed at the University of California, Berkeley, laboratory of Jennifer Doudna, co-inventor of CRISPR-Cas9 genome editing, involves wrapping Cas9 editing proteins and guide RNAs in a membrane bubble decorated with pieces of monoclonal antibodies that target specific types of blood cells. As a demonstration, the researcher targeted a cell of the immune system, a T lymphocyte, which is the starting point for a revolutionary cancer treatment called CAR- T cell therapy. The research team treated live mice equipped with a humanized immune system and transformed their human T cells into CAR T cells capable of targeting and eliminating another class of immune cells, the B cells. 

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies.  The authors of this study have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. In this work, to simplify the vector design, they decided to codon-optimize the entire RD114-TR ORF. In fact, the elimination of the interfering sequences would have avoided using the BGI, therefore reducing the size of the vector. Unexpectedly, they found that, despite the high level of transcription/translation and cytosol export, RD114-TRco is functionally dead. These data strengthen the conclusion, also supported by other studies, that codon optimization may not always lead to functional improvement of the gene of interest.

Research into the properties of Japanese encephalitis virus (JEV) has been facilitated by use of pseudotyped viruses. In this study, the scientists generated three lentiviral vectors pseudotyped with JEV envelope proteins that co-express either a strong signal peptide from vesicular stomatitis virus (VSV)-G (VSVMEpv) or a weak signal peptide of JEV (SPMEpv), or a virus without a signal peptide in front of the JEV prM/E (MEpv).

Their results show that the titer of VSVMEpv was 17-fold higher than that of MEpv, while the titer of SPMEpv was six-fold higher than that of MEpv.  

This study provided a cost-effective method to generate pseudotyped JEV-E based lentiviral vectors, which may represent a valid model to investigate some of the infectious properties of JEV.

In this work, the authors tested whether known viral pseudo-typing proteins or surface proteins known to be recruited to the HIV envelope could be engineered to carry neutralising epitopes from another microorganism onto the lentiviral surface. Their results identify ICAM1 as a novel vehicle for lentiviral pseudo-typing. Importantly, they show that in a model lentiviral system ICAM1 can be engineered in chimeric form to result in expression of a fragment of the Tetanus toxoid on the viral membrane and that these viruses can then be neutralised by human serum antibodies protective against Tetanus. This raises the possibility of delivering chimeric antigens as a gene therapy in HIV infected patients.

Naldini et al. developed a shielded lentiviral vector (LV) able to escape phagocytosis by increasing the content of the phagocytosis inhibitor CD47 on their surface. Upon intravenous administration in monkeys, the LVs showed high transduction efficacy without signs of toxicity. The results suggest that LV-mediated gene therapy might be an effective strategy for treating hemophilia and possibly other disorders.

Here, the scientists report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra.

In order to develop novel strategies to genetically manipulate T-ALL leukaemia-initiating cells (LIC), the scientists examined whether primary TALL cells express the receptors for different lentiviral vector pseudotyping glycoproteins, such as the vesicular-stomatitis-virus G protein (VSVG) (4), the measles virus hemagglutinin (H) and fusion (F) glycoproteins (5) and the baboon retroviral envelope glycoprotein (BaEV) (6). In summary, they find that BaEV-pseudotyped LVs have an exceptional capacity to transduce T-ALL LICs without altering their self-renewing properties.

B cells are attractive targets for gene therapy of diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. However, these cells are commonly refractory to transducing gene vectors. In this study, the scientists design a new lentiviral vector with the baboon envelope. They tested this new vector for human (h) B-cell transduction followed their adaptive transfer into NSG mouse. They observe that, upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, while VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. As proof of principle, they assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo.

This study illustrates the multifaceted feature of lentiviral vectors that makes it a high powerful gene transfer tool.

GEG Tech offer high efficient lentiviral vectors with several different design options, including different pseudotype. We provide VSV or Mokola envelope pseudotyped lentiviral vectors through our catalog and design also other envelopes on demand.

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In mammals, interferon-inducible transmembrane proteins (IFITMs) prevent infections by various enveloped viruses.

Here the authors show that he knockdown of IFITM3 in DF-1 cells by siRNA increased the infectivity of a vesicular stomatitis virus G protein-pseudotyped lentiviral vector, suggesting that lower levels of IFITM3 are still sufficient to restrict this viral vector.