Biologics by design

  • Nanoplasmids™

    Nanoplasmids™

    NTC's newest and best-in-class vectors

  • RNA-OUT, Viral Vector/Transposon Nanoplasmid™ Retrofits

    Viral Vector/Transposon Nanoplasmid™ Retrofits

    NTC’s antibiotic free Nanoplasmids™ are easily substituted for conventional plasmids, minicircles, doggy bones, etc., for use with all major therapeutic vector groups. The Nanoplasmid™ backbone has profound and unexpected beneficial effects on performance (expression levels and duration). The Nanoplasmid™ is now available for use in: Lenti vectors, CAR-T cell vectors, AAV, transposons, etc. NTC’s RNA-OUT sucrose selection is a regulatory agency preferred antibiotic-free backbone, and has already been approved and used successfully in a number of first-in-human clinical trials.

    NTC now offers its Nanoplasmid™ Retrofit services, which includes verification and DNA production. Send us 10 micrograms of your plasmid (on filter paper or in liquid form) and NTC will: swap your current plasmid backbone for the Nanoplasmid one, verify the construct, and return the desired quantity of the amplified product, along with the R6K host strain. Its small size also increases potency and reduces the cost of producing plasmid copy number equivalents.

     

     

    In summary, the Nanoplasmid (RNA-OUT selectable marker plus R6K origin) replaces antibiotics with sucrose selection, and replaces the promiscuous pUC origin with the host-restricted R6K one. It is:

     

    • Regulatory agency compliant,

     

    • Smaller in size (452bp) than conventional marker/ORIs, therefore increasing potency and decreasing material cost,

     

    • Easily swappable into most types of viral and nonviral vectors,

     

    • Grams per liter fermentation, not requiring post-production modification, and therefore ideal for vector products that are destined for clinical use, and

     

    • Dramatically improves expression levels and duration of expression compared to Kanamycin vectors.

  • Mammalian Expression Vectors

    Enhanced recombinant protein expression in mammalian cells and tissues: superior genetic immunization

    NTC '8' Series Mammalian Expression Vectors

    NTC's PDF Library

    A typical advanced NTC vector, NTC8685-EGFP, with the following features: markerless (sucrose selection), enhanced transcription (SV40/CMV), enhanced translation (CMV/HTLV-1-R), enhanced translation (VA1), enhanced plasmid production (PAS-BH).

    NTC Vector Advantages

     

    • DNA Vaccination

     

    • Therapeutic Vector

     

    • Native Expression

     

    • Secreted (TPA tag)

     

    • Minimal Backbone

     

    • Reporter (EGFP)

     

    • Regulatory Compliance

     

    • Simultaneous Cloning

  • Antibiotic Free Vectors

    Antibiotic Free Vectors

    NTC's PDF Library

    Antibiotic-Free RNA-OUT Marker for Mammalian Expression and DNA Vaccine Vectors

    Antibiotic Free Vectors, Antisense RNA-OUT marker for plasmid selection

    NTC Vector Advantages:

    • DNA Vaccination

    • Therapeutic Vector

    • Native Expression

    • Secreted (TPA tag)

    • Minimal Backbone

    • Reporter (EGFP)

    • Regulatory Compliance

    • Simultaneous Cloning

    Features:
    • Replaces KanR resistance gene with antisense RNA-OUT, 150bp RNA marker
    • Kit includes strain for selection with RNA-OUT
    • Enhanced expression with optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)
    • Complies with regulatory guidance
    • Increased vaccine potency and safety by replacement of KanR gene
    • Improved DNA backbone for maximum plasmid production in
    E. coli.

    NTC offers a retrofitting service, directly replacing the Kan gene in your current vectors with the RNA-OUT cassette. Natx.com/cloning_qc_retrofit.html

    In keeping with regulatory agency guidance recommending removal of antibiotic resistance genes from vectors used in humans, NTC developed its leading antibiotic free selection system (Fig.1) based on sucrose. The KanR gene is replaced with a 150bp RNA-OUT antisense RNA, which represses expression of the (SacB) gene, located in the host chromosome. This innovation has the additional advantages of: shrinking the plasmid backbone (making it more potent); and decreasing the cost of production through high yield replication in E. coli.

  • Rig-I Activating Vectors

    Rig-I Activating Vectors

    NTC's PDF Library

    RIG-I Activating Vectors: NTC8685-eRNA41H-EGFP

    Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza DNA Vaccine, Luke, J.M., Simon, G.G., Soderholm, J., Errett, J.S., August, J.T., Gale, J., Hodgson, C.P., Williams, J.A. J. Virol. 85:1370-83, (2011).

    Features

    • Enhanced antigen expression in mammalian cells

    • Compliance with FDA guidance

    • Improved antibody response

    • Improved antibody avidity

    • Kanamycin or RNA-OUT (antibiotic free) selectable markers

    • Improved DNA backbone for maximum plasmid production (>2.6g/L)

    • Enhanced co-stimulatory RNAe™ activates RIG-1 pathway, Type I interferon production

    • Optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)

    • Superior E. coli plasmid production yields (>2.6g/L)

    • Optional TPA secretion tag

    Rig-I Activation on IFNβ Promoter Luciferase

    Methods to improve DNA vaccine-induced adaptive immunity are needed.  This may be accomplished using plasmid vector backbone-encoded innate immunity agonists. Retinoic-acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (mda5) are critical cytoplasmic double stranded RNA (dsRNA) pattern receptors required for innate immune activation in response to viral infection. Activation of RIG-I and mda5 leads to type I interferon (IFN) and inflammatory cytokine production through IPS-1 activation. Since type I interferon enhances antigen-specific immune responses, we hypothesized that DNA vaccines coexpressing a RIG-I RNA agonist (eRNA) would generate superior immune responses to the encoded antigen. Indeed, expression of  RIG-I activating immunostimulatory RNA from the vector backbone (Fig 1) may be used to activate innate immunity and improve adaptive immune responses (Luke et al., 2011).

     

    Improved Innate and Adaptive Immune Responses

     

  • dcm- Strains

    dcm- Strains

    NTC's PDF Library

    For easy purification of recombinant proteins and plasmid DNA

    NTC8685 is a highly effective, optimized DNA plasmid vector with sucrose selectable marker.

    Features:

     

    • Standard E. coli production host strains (DH5a, XL1-blue) modified to eliminate dcm methylation of plasmid DNA

     

    • Kanamycin-resistance or antibiotic-free (RNA-OUT) selection vectors (for 7-series or 8-series vectors, respectively)

     

    • Gram-plus per liter yields possible with KanR or antibiotic free (sucrose selection) strains

     

    • Increased expression in eukaryotic cells, combined with decreased immunogenicity, for gene therapy applications

     

    • Can be used with non-NTC Kanamycin-resistance vectors (use strain NTC48107 only)

    NTC7482-41H-VA2-HA plasmid (HA transgene version of NTC7482-41H-VA2-EGFP). Average_SD anti-HA2 total IgG, IgG1, and IgG2a titers at the indicated serum dilutions after prime-boost immunization of naked dcm+ (+) or dcm-(-) plasmid. The total IgG and IgG2a dcm+ versus dcm- titers were significantly different at a Pvalue 0.05 [Wilcoxon (Mann–Whitney) rank-sum test].

    •  Enhanced Gene expression

    •  Decreased immuno-genicity (for therapeutic, vs. vaccine use)

    •  Derivatives of well-known strains: DH5a and XL1-blue

    •  Can be used with standard antibiotic resistance markers

    The dcm methylase recognizes the internal cytosine residues in the recognition sequence 5'-CC*AGG-3' or 5'-CC*TGG-3'. This creates 5-methyl-cytosine (5mC), a common mammalian pattern (CG methylation) although the dcm methylated cytosine is in a different sequence context in bacteria. While plasmid production yields and quality are similar between dcm+ and dcm- host strains, CMV promoter expression is reduced by dcm methylation. Surprisingly, despite improved expression, dcm- plasmid DNA is less immunogenic. These results suggest  that, while dcm+ strains are optimal for DNA vaccination, that dcm- strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. (Carnes et al. 2010, Biotechnol. Bioeng.).

     

    While DH5a is the production host of choice for most plasmid vectors, XL1Blue is a superior host for production of small (<3 kb) vectors (Carnes et al. 2010, J. Gene Med.). NTC offers dcm+ and dcm- versions of these cell lines for vaccine (dcm+) or therapy (dcm-) gene medicine plasmid production.

     

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