Genetic Engineering Publications - GEG Tech top picks

A new approach to CRISPR-Cas9 technology allows scientists to introduce particularly long DNA sequences at precise locations in cell genomes at remarkably high efficiencies without the viral delivery systems traditionally used to transport DNA into cells. The researchers found that high levels of double-stranded DNA template can be toxic to cells, resulting in low efficiency. The team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations. The researchers therefore describe in the paper published in the journal Nature Biotechnology, a method for attaching the modified Cas9 enzyme to a single-stranded template DNA, by adding just a small overhang of double-stranded DNA at the ends. The single-stranded template DNA could more than double the efficiency of gene editing compared to the old double-stranded approach. In addition, the double-stranded ends of the molecules allow the researchers to use Cas9 to enhance the delivery of non-viral vectors into cells. In the study, the researchers used the new DNA template to generate more than one billion CAR-T cells targeting multiple myeloma and showed that their approach could, for the first time, replace two genes associated with rare genetic immune diseases, IL2RA and CTLA4, in their entirety

A new approach to CAR T cells therapy developed by a UCLA research team allows for an effective and long-lasting response to HIV, unlike current applications of such therapies that cannot provide lasting immunity, i.e., they cannot destroy malignant or infected cells that appear months or years after treatment. Previously, researchers had created a therapy based on CAR T cells containing part of the CD4 molecule because HIV binds to CD4 molecules in order to infect cells in the body. HIV binds to the CD4 molecule of the CAR T cell, which activates it and the cell kills HIV. However, two domains of CD4 molecules still allowed HIV to infect cells. So, the researchers removed these domains and added another that makes the cells resistant to infection and allows a more effective and durable cellular response against HIV than the former therapy. This new CAR T cells therapy with a truncated form of the CD4 molecule works like a vaccine. It stimulates the patient's immune system before HIV itself induces a response. In addition, this therapy induces the production of a large number of memory T cells and could influence the field of immunotherapy focused on engineering T cells with CAR molecules.

To restore dystrophin production and myocyte function in patients with Duchenne muscular dystrophy (DMD), several CRISPR strategies have been tried to correct some of the hundreds of thousands of documented mutations in the dystrophin gene (DMD). Among these strategies, researchers at the University of Texas Southwestern Medical Center in Dallas, Texas, have developed a novel CRISPR gene editing strategy for DMD therapy. This was tested in animal and human DMD models where the researchers deleted exon 51 in the DMD gene which disrupted the dystrophin reading frame and generated a premature stop codon in exon 52. The researchers were able to restore the reading frame with both basic and main editing by introducing exon skipping and reframing, respectively, through their "single-swap" ABE strategy, i.e., through editing a single letter in the splice acceptor or donor site can cause exon skipping. The researchers targeted the splice acceptor or splice donor site of exon 50 or 52 and the single nucleotide modification restored dystrophin expression in human cardiomyocytes and contractile function was normalized using master editing. However, one challenge is the high doses required to deliver expression throughout the body.