Production of lentiviral vectors using novel, enzymatically-produced, linear DNA vectors

The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses ‘rolling-circle’ amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular injection. Luciferase expression was quantified in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification

Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy

In vivo effects of interferon-Γ and anti-interferon-Γ antibody on the experimentally induced lichenoid tissue reaction

We investigated the in vivo effect of recombinant interferon-Γ (IFN-Γ) and tumour necrosis factor Α (TNF-Α) treatment of mice on the development of the delayed-type hypersensitivity (DTH) reaction and lichenoid tissue reaction (LTR) following the local injection of cloned autoreactive T cells. Both the DTH reaction and the LTR were significantly enhanced by pre-treatment with IFN-Γ, but not with TNF-Ã. Induction of class II MHC antigens on keratinocytes was not essential for the enhancement by IFN-Γ. Administration of anti-IFN-Γ antibody reduced the DTH reaction and LTR, although complete inhibition was not observed with our treatment regimen. The ability of IFN-Γ to increase the number of the cloned T cells invading the epidermis in vivo , is in keeping with our previous observation that IFN-Γ treatment of cultured keratinocytes markedly increased the adherence reaction between T cells and keratinocytes in vitro.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74579/1/j.1365-2133.1988.tb03202.x.pd

Elevation in Body Temperature to Fever Range Enhances and Prolongs Subsequent Responsiveness of Macrophages to Endotoxin Challenge

Macrophages are often considered the sentries in innate immunity, sounding early immunological alarms, a function which speeds the response to infection. Compared to the large volume of studies on regulation of macrophage function by pathogens or cytokines, relatively little attention has been devoted to the role of physical parameters such as temperature. Given that temperature is elevated during fever, a long-recognized cardinal feature of inflammation, it is possible that macrophage function is responsive to thermal signals. To explore this idea, we used LPS to model an aseptic endotoxin-induced inflammatory response in BALB/c mice and found that raising mouse body temperature by mild external heat treatment significantly enhances subsequent LPS-induced release of TNF-α into the peritoneal fluid. It also reprograms macrophages, resulting in sustained subsequent responsiveness to LPS, i.e., this treatment reduces “endotoxin tolerance” in vitro and in vivo. At the molecular level, elevating body temperature of mice results in a increase in LPS-induced downstream signaling including enhanced phosphorylation of IKK and IκB, NF-κB nuclear translocation and binding to the TNF-α promoter in macrophages upon secondary stimulation. Mild heat treatment also induces expression of HSP70 and use of HSP70 inhibitors (KNK437 or Pifithrin-µ) largely abrogates the ability of the thermal treatment to enhance TNF-α, suggesting that the induction of HSP70 is important for mediation of thermal effects on macrophage function. Collectively, these results support the idea that there has been integration between the evolution of body temperature regulation and macrophage function that could help to explain the known survival benefits of fever in organisms following infection

The invasin of Yersinia spp. provides co-stimulatory activity to human T-cells through interaction with beta1- integrins

The invasin proteins of Yersinia spp. are outer membrane proteins which are involved in the penetration of these bacteria into mammalian cells (Cell 1990. 60: 861). Invasin binds to several different beta1 integrins with extremely high affinity, the integrin-binding domain of invasin has been mapped to the C-terminal 192 amino-acids of the molecule (J. Biol. Chem. 1991. 266: 24367). Expression of this fragment alone on the cell surface of non-invasive bacteria is enough to confer the invasive phenotype on these strains (EMBO J. 1990. 9: 1979). Here we show that the carboxy-terminal 192 amino acids of invasin expressed as a fusion protein with the maltose binding protein of E. coli is capable of delivering co-stimulatory signals to human T cells through the beta1 integrins. Co-stimulation was assayed by the ability of invasin to augment the response of highly purified CD4+ and CD8+ T cells to co-immobilized anti-CD3 antibody. Antibody blocking studies indicated that the co-stimulation was mediated through beta1 integrins. The proliferation induced by co-stimulation of CD4+ T cells was accompanied by the synthesis of the cytokines tumor necrosis factor-alpha and interferon-gamma, whereas the activation of CD8+ T cells led to the generation of cytotoxic effectors. The region of the invasin molecule involved in T cell activation was further mapped using synthetic peptides. A region of the invasin molecule containing the residues TAKSKKFPSY could substitute for invasin in T cell activation. The co-stimulation by peptide could also be inhibited by anti-integrin antibodies. The observation that an outer membrane protein of a bacterium which is associated with reactive arthritis and other autoimmune spondyloarthropathies can act as a T cell co-stimulus may have implications for the etiology of these diseases

Recombinant P.69/pertactin: immunogenicity and protection of mice against Bordetella pertussis infection.

The immunogenicity of recombinant (r-) pertactin was examined. Parenteral immunization of mice with natural or r-pertactin produced a similar increase in serum anti-pertactin antibodies and a decrease in Bordetella pertussis lung counts following aerosol challenge. Study of the kinetics of B. pertussis growth in the respiratory tract of immunized and control mice revealed that immunization with r-pertactin halted the multiplication of B. pertussis in the lungs and facilitated the early onset of bacterial clearance. In the trachea, bacterial numbers declined sharply in immunized animals during the first 3 days after challenge but thereafter B. pertussis numbers remained fairly constant throughout the rest of the experiment. Very low doses (0.1 micrograms) of r-pertactin were immunogenic and protective but only if the antigen was absorbed to alhydrogel. In vitro proliferation assays with lymphocytes from mice primed with either natural or r-pertactin indicated that the major T-cell epitopes of pertactin are conserved in the recombinant protein

Expression of L-selectin on Th1 cells is regulated by IL-12.

L-selectin has become established as a key molecule in the recirculation of naïve T cells from the blood to peripheral lymph nodes, yet little is known about its role in the migration of effector or memory cells. While differentiating naïve CD4+ T cells into Th1 and Th2 subsets in vitro, it was noted that L-selectin levels were maintained on the Th1 subset of cells. The expression of L-selectin on the Th1 cells appeared to be dependent on the presence of IL-12. Th2 cells, differentiated in the absence of IL-12, failed to maintain L-selectin expression. Coculture with IL-12, IL-18, IL-4, TNF-alpha, or IFN-alpha, -beta, or -gamma demonstrated a dependence on IL-12 alone for L-selectin expression. In addition, the inclusion of heat-killed Listeria monocytogenes in the cultures also maintained L-selectin expression on the Th1 cells. In all cultures, the maintenance of L-selectin on the T cell surface could be blocked by the inclusion of anti-IL-12 Abs. Analysis of the mRNA levels for L-selectin in T cells, differentiated in the presence or absence of IL-12, showed that the cytokine appears to exert its effect on L-selectin at the transcriptional level. Given the key role played by IL-12 in the differentiation of naïve T cells into the Th1 subset, the observation that IL-12 can also regulate L-selectin expression has implications for the migration of Th1 effector cells both through the lymphatic system and to sites of inflammation