Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.
Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity.Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group-and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and the preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.
The development of new therapies to slow-down or halt Parkinson's disease progression is a healthcare priority. A key pathological feature is the presence of alpha-synuclein aggregates and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently the down-regulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential and siRNAs specific to alphasynuclein have been designed, however the efficacy of siRNA treatment is limited by its shortterm efficacy. To combat this we designed shRNA minicircles (shRNA-MC), with the potential for prolonged effectiveness, and used RVG-exosomes as vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing green fluorescent protein (GFP) and demonstrated its ability to down-regulate GFP protein expression in the brain for up to 6 weeks. RVG exosomes were used to deliver anti alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed fibrils induced model of parkinsonism. This therapy decreased alphasynuclein aggregation, reduced the loss of dopaminergic neurones and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MC delivered by RVGexosomes for long-term treatment of neurodegenerative diseases.
A large number of cancer gene therapy clinical trials are currently being performed that are attempting to evaluate novel approaches to eliminate tumor cells by the introduction of genetic material into patients. One of the most important objectives in gene therapy is the development of highly safe and efficient vector systems for gene transfer in eukaryotic cells. Currently, viral and nonviral vector systems are used, both having their advantages and limitations. Minicircles are novel supercoiled minimal expression cassettes, derived from conventional plasmid DNA by site-specific recombination in vivo in Escherichia coli for the use in nonviral gene therapy and vaccination. Minicircle DNA lacks the bacterial backbone sequence consisting of an antibiotic resistance gene, an origin of replication, and inflammatory sequences intrinsic to bacterial DNA. In addition to their improved safety profile, minicircles have been shown to greatly increase the efficiency oftransgene expression in various in vitro and in vivo studies. In this chapter, we describe the production, purification, and application of minicircle DNA and discuss the rationale of the improved gene transfer efficiencies compared to conventional plasmid DNA.
The results obtained in the present study demonstrate that the described technology facilitates the production of highly pure minicircle-DNA for direct application in gene therapy and vaccination. The process described is efficient, stable and suitable for further scale-up in industrial large-scale manufacturing.
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