Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Terminal organ weights (mg wet tissue) and body weights (grams) in female and male NPC1 −/− mice treated with either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: H&E stains of cerebral cortex ( A – C ), spleen ( D – F ), and liver ( G – I ) from NPC1 −/− mice or control mice at euthanasia. Panels ( A , D , G ) are NPC1 −/− mice treated with vehicle; panels ( B , E , H ) are NPC −/− mice treated with TfRMAb/pPDGFB-NPC1 THLs. Panels ( C , F , I ) are control mice. Arrows in panels ( A , D , G ) point to cholesterol-laden cells in brain, spleen, and liver, respectively. Brain sections are through cortical layers I–IV. Magnification is the same for all panels and the panel A magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Control

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Electron microscopy of cerebral cortex of vehicle ( A , B ) or THL ( C , D ) treated NPC1 −/− mouse at euthanasia. Multi-lamellated vacuoles of varying size are present in the cytoplasm of brain cells. Magnification in panels ( A ) and ( C ) is the same and magnification bar in panel ( A ) is 1,000 nm; magnification in panels ( B ) and D is the same and magnification bar in panel ( B ) is 1,000 nm.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: GFAP immunocytochemistry of thalamus of NPC1 −/− mouse treated with vehicle ( A ) or the TfRMAb/pPDGFB-NPC1 THLs ( B ). The GFAP immunocytochemistry of thalamus from a control mouse is shown in ( C ). Sections are counter-stained with hematoxylin. Magnification of all 3 panels is the same and the panel ( A ) magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Immunocytochemistry, Control, Staining

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ concentrations (pg plasmid DNA per mg wet tissue) in female and male NPC1 −/− mice at 4 days following the last IV injection of either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Plasmid Preparation, IV Injection

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ enrichment of NPC1 mRNA, relative to GAPDH mRNA, in THL treated NPC1 −/− mice as compared to vehicle treated NPC1 −/− mice.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: NPC1 Western blot of the spleen of 3 vehicle treated NPC −/− mice and 3 THL treated NPC −/− mice. The spleens of the THL treated mice express the 200 kDa immunoreactive NPC1 protein.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Western Blot

Journal: Cell host & microbe

Article Title: Structural Basis for a Convergent Immune Response against Ebola Virus.

doi: 10.1016/j.chom.2020.01.007

Figure Lengend Snippet: Figure 2. VH3-15/Vl1-40 mAbs Target a Partially Conserved Epitope on GP (A) Close-up view of the epitope on EBOV GP (magenta) that is recognized by VH3-15/Vl1-40 mAbs, exemplified with 5T0180 (orange and yellow surface representations for the heavy and light chains, respectively). The different patches that make the recognition site are colored in blue, green, red, and gray. The main residues on GP that make the epitope are labeled. (B) Multiple sequence alignments of EBOV, SUDV, and BDBV (accession codes NP_066246, YP_138523, and YP_003815435, respectively). Residue numbering is of EBOV. The relevant parts that make the epitope on EBOV GP are labeled and highlighted using the same colors as in (A). (C) Footprints of the three VH3-15/Vl1-40 mAbs. The buried surface areas are shown on a single EBOV GP1 protomer in different colors for each of the mAbs. The calculated buried solvent accessible areas upon complex formation are indicated for each mAb. (D) VH3-15/Vl1-40 mAbs sterically prevent NPC1 binding. Superimposition of a single EBOV GP/5T0180 protomer (orange, yellow, and violet for the heavy chain, light chain, and GP, respectively) with the model of NPC1 bound to EBOV GP (PDB: 5F1B, blue—showing NPC1 only). 5T0180 and NPC1 occupy the same volume and, hence, cannot bind at the same time. (E) VH3-15/Vl1-40 mAbs retain binding to EBOV GPcleaved in acidic pH. This plot is showing single cycle kinetic experiments at pH 5.5 using EBOV GPcleaved as analyte that was injected at the indicated concentrations over mAbs 1T0227 (blue curve) and 5T0180 (red curve) that were conjugated to a sensor chip. Since this configuration enables high avidity (three potential binding sites per trimer), we did not attempt to derive kinetic parameters. (F) 1T0227 and 5T0180 compete with NPC1 for binding to EBOV GPcleaved. EBOVcleaved (50 nM) was injected over immobilized NPC1 at pH 5.5 alone (blue curve) or in the presence of 500nM mAbs 1T0227, 5T0180, or 3T0331 (anti-GP2 mAb, serves as a negative control). See also Figure S2.

Article Snippet: For cloning of fusion protein NPC1-Fc, residues 374-620 (domain C) of mouse NPC1 (a kind gif of Matthew Scott (Lopez et al., 2011), Addgene vector #53521) were cloned upstream to a human Fc region derived from IgG1 and downstream a signal peptide using BamHI / KpnI restriction sites as previously described (Cohen-Dvashi et al., 2018)

Techniques: Labeling, Sequencing, Residue, Solvent, Binding Assay, Injection, Negative Control

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Terminal organ weights (mg wet tissue) and body weights (grams) in female and male NPC1 −/− mice treated with either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: H&E stains of cerebral cortex ( A – C ), spleen ( D – F ), and liver ( G – I ) from NPC1 −/− mice or control mice at euthanasia. Panels ( A , D , G ) are NPC1 −/− mice treated with vehicle; panels ( B , E , H ) are NPC −/− mice treated with TfRMAb/pPDGFB-NPC1 THLs. Panels ( C , F , I ) are control mice. Arrows in panels ( A , D , G ) point to cholesterol-laden cells in brain, spleen, and liver, respectively. Brain sections are through cortical layers I–IV. Magnification is the same for all panels and the panel A magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques: Control

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Electron microscopy of cerebral cortex of vehicle ( A , B ) or THL ( C , D ) treated NPC1 −/− mouse at euthanasia. Multi-lamellated vacuoles of varying size are present in the cytoplasm of brain cells. Magnification in panels ( A ) and ( C ) is the same and magnification bar in panel ( A ) is 1,000 nm; magnification in panels ( B ) and D is the same and magnification bar in panel ( B ) is 1,000 nm.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: GFAP immunocytochemistry of thalamus of NPC1 −/− mouse treated with vehicle ( A ) or the TfRMAb/pPDGFB-NPC1 THLs ( B ). The GFAP immunocytochemistry of thalamus from a control mouse is shown in ( C ). Sections are counter-stained with hematoxylin. Magnification of all 3 panels is the same and the panel ( A ) magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques: Immunocytochemistry, Control, Staining

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ concentrations (pg plasmid DNA per mg wet tissue) in female and male NPC1 −/− mice at 4 days following the last IV injection of either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques: Plasmid Preparation, IV Injection

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ enrichment of NPC1 mRNA, relative to GAPDH mRNA, in THL treated NPC1 −/− mice as compared to vehicle treated NPC1 −/− mice.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: NPC1 Western blot of the spleen of 3 vehicle treated NPC −/− mice and 3 THL treated NPC −/− mice. The spleens of the THL treated mice express the 200 kDa immunoreactive NPC1 protein.

Article Snippet: A human NPC1 expression plasmid, under the influence of the human PDGF-B promoter, was custom synthesized as a 5.6 kb expression cassette, which was subcloned in a 8.0 kb pUC plasmid DNA at Genscript (Piscataway, NJ), and the sequence of the entire expression cassette was confirmed by bidirectional DNA sequencing.

Techniques: Western Blot