Journal: PLoS ONE

Article Title: Improved targeting of human CD4 + T cells by nanobody-modified AAV2 gene therapy vectors

doi: 10.1371/journal.pone.0261269

Figure Lengend Snippet: (A) Amino acid alignment of the Nb sequences used in this study. Nb annotation is in accordance with the study by Traenkle and coworkers , which describes thorough Nb characterization and application for in vivo imaging. Of note, CD4-Nb1a is not specified in Traenkle et al., as its complementary determining region 3 (CDR3, black box) is identical to CD4-Nb1 (amino acids 101–117 in the alignment). (B) Plasmid constructs used for production of indicated AAV2 particles and schematic representation of the resulting capsids. Equimolar amounts of plasmids were co-transfected into HEK293T cells (see ). For clarity, adenoviral helper plasmid and vector encoding the eGFP reporter included in all co-transfections are not depicted. Essential changes to optimized viral capsid proteins VP1-3 are indicated: red = mutated / black = wt. Disabled open reading frames are greyed out. ATG = start codon; SA = splice acceptor site; Nb = full length nanobody sequence as presented in A (orange); L = glycine-serine linker genetically fusing nanobody monomers in the bivalent constructs (pink); R585A/R588A = Arg to Ala amino acid substitution at residues 585 and 588 (blind). AAV capsids were rendered with help of the browser tool by Antonio Negrón .

Article Snippet: In all co-transfection reactions, the helper plasmid pAdDeltaF6 (Addgene #112867) and the AAV2 vector genome pscAAV-CAG-GFP (Addgene #83279) encoding the GFP transgene under a constitutive CAG promoter were included.

Techniques: In Vivo Imaging, Plasmid Preparation, Construct, Transfection, Sequencing

Journal: PLoS ONE

Article Title: Improved targeting of human CD4 + T cells by nanobody-modified AAV2 gene therapy vectors

doi: 10.1371/journal.pone.0261269

Figure Lengend Snippet: (A) Purified AAV2 particles were subjected to negative staining electron microscopy. Representative images show vector genome-containing (black arrow) and empty (white arrow) capsids. Individual capsids are magnified. Scale bar 50 nm. (B) Immuno-gold staining of purified AAV2 particles indicate accessible Nbs at the capsid surface. Images were cropped to show representative capsids. Scale bar 100 nm. (C) Western blot of purified vector particles using a VP1/VP2-specific antibody (A69). Individual bands are labeled; HSPG-ko indicate shifted protein bands compared to opt AAV2 due to R585/588A heparan sulfate proteoglycan (HSPG) blinding. (D) Capsid thermal stability assay of purified vector particles using AAV-specific antibody A20 detecting intact capsids. Temperature gradient is indicated.

Article Snippet: In all co-transfection reactions, the helper plasmid pAdDeltaF6 (Addgene #112867) and the AAV2 vector genome pscAAV-CAG-GFP (Addgene #83279) encoding the GFP transgene under a constitutive CAG promoter were included.

Techniques: Purification, Negative Staining, Electron Microscopy, Plasmid Preparation, Staining, Western Blot, Labeling, Stability Assay

Journal: PLoS ONE

Article Title: Improved targeting of human CD4 + T cells by nanobody-modified AAV2 gene therapy vectors

doi: 10.1371/journal.pone.0261269

Figure Lengend Snippet: (A) CD4 expression on cell lines used in this study. Cells were stained with anti-CD4-Cy7 antibody and compared to unstained mock controls. The CD4 signal in mock cells was manually adjusted to 100 arbitrary units and compared with stained cells by flow cytometry. Histograms were normalized by modal mode to visualize comparable peak heights. (B) Two HeLa (wt and TZMbl) and two T lymphoid (1G5 and SupT1) cell lines were transduced with the indicated viral vector genome copies per cell (gc/cell). Three days post infection the % of eGFP positive cells was determined by flow cytometry; n = 2–4. Error bars indicate the SD. (C) 1G5 cells were transduced with the indicated AAV2 particles at a concentration of 10,000 gc/cell. Three days post transduction cells were stained for CD4 expression and analyzed for GFP expression by flow cytometry. Representative data are shown.

Article Snippet: In all co-transfection reactions, the helper plasmid pAdDeltaF6 (Addgene #112867) and the AAV2 vector genome pscAAV-CAG-GFP (Addgene #83279) encoding the GFP transgene under a constitutive CAG promoter were included.

Techniques: Expressing, Staining, Flow Cytometry, Transduction, Plasmid Preparation, Infection, Concentration Assay

Journal: PLoS ONE

Article Title: Improved targeting of human CD4 + T cells by nanobody-modified AAV2 gene therapy vectors

doi: 10.1371/journal.pone.0261269

Figure Lengend Snippet: (A) Representative analysis of a mixed culture experiment. HeLa wt (CD4 negative) were mixed with HeLa TZMbl (CD4 positive) in a ratio of 1:1 prior to AAV2 transduction and subsequently transduced with different virus dilutions. Three days post transduction, cells were harvested, stained for CD4 and analyzed for GFP expression by flow cytometry. (B) Cumulative data from independent HeLa mixed culture experiments. Indicated are the relative frequencies of GFP positive cells for CD4 positive and negative cells (on the left). AAV2 CD4-specific transduction is calculated as ratio from the individual cell populations (fold CD4 positive over CD4 negative). Fold changes are indicated (on the right); n = 2–6, presented are means with SD, significant differences indicated with asterisks: * p < .05, ** p < .01, *** p < .001, **** p < .0001.

Article Snippet: In all co-transfection reactions, the helper plasmid pAdDeltaF6 (Addgene #112867) and the AAV2 vector genome pscAAV-CAG-GFP (Addgene #83279) encoding the GFP transgene under a constitutive CAG promoter were included.

Techniques: Transduction, Virus, Staining, Expressing, Flow Cytometry

Journal: Cell

Article Title: An Excitatory Circuit in the Perioculomotor Midbrain for Non-REM Sleep Control.

doi: 10.1016/j.cell.2019.03.041

Figure Lengend Snippet: Figure 4. Glutamatergic but Not Cholinergic pIII Neurons Promote NREM Sleep (A) Overlap between Calca and Slc17a6 (top row) or Chat (bottom row) in the pIII region, measured by double FISH. Shown are coronal images at AP 3.8 mm. Arrowheads indicate co-labeled cells. Blue, DAPI. Scale bar, 100 mm. Right: percentages of Calca, Slc17a6, or Chat neurons that are co-labeled. Each circle indicates data from one mouse (n = 5 mice). (B) Optogenetic activation of cholinergic pIII neurons did not affect sleep. Left: fluorescence image of the pIII region (red box in the coronal diagram at AP 3.8 mm) in a ChatCre mouse injected with AAV-EF1a-DIO-hChR2-eYFP. Blue, DAPI. Scale bar, 100 mm. Right: percentage of time in the NREM, REM, or wake state before, during, and after laser stimulation, averaged from 4 mice. Shading, 95% CI. Blue stripe, laser stimulation period (20 Hz, 120 s).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-GFP for immunohistochemistry Aves Labs GFP-1020; RRID: AB_10000240 anti-Fos Santa Cruz sc-52; RRID:AB_2106783 anti-GFP for immunoprecipitation Memorial-Sloan Kettering Monoclonal Antibody Facility Htz-GFP-19F7 and Htz-GFP-19C8; RRID: AB_2716736 and AB_2716737 Bacterial and Virus Strains AAV2-EF1a-DIO-ChR2-eYFP UNC Chapel Hill Vector Core NA AAVDJ-EF1a-DIO-ChR2-eYFP Stanford University Virus Core NA AAV2-EF1a-DIO-iC++-eYFP UNC Chapel Hill Vector Core NA AAV2-EF1a-DIO-eYFP UNC Chapel Hill Vector Core NA AAV2-EF1a-DIO-hM3D(Gq)-mCherry UNC Chapel Hill Vector Core NA AAV2-EF1a-DIO-hM4D(Gi)-mCherry UNC Chapel Hill Vector Core NA AAV2-EF1a-DIO-mCherry UNC Chapel Hill Vector Core NA AAV2-EF1a-DIO-taCasp3-TEVp UNC Chapel Hill Vector Core NA AAV2-CAG-FLEx-TC66T Addgene 48331 AAV2-CAG-FLEx-RG Addgene 74292 rAAV2-retro-EF1a-DIO-mCherry This paper Tervo et al., 2016 rAAV2-retro-EF1a-DIO-eGFP This paper Tervo et al., 2016 RG-deleted, eGFP-expressing rabies virus Gene Transfer Targeting and Therapeutics Core of Salk Institute NA Chemicals, Peptides, and Recombinant Proteins Clozapine-N-oxide Sigma-aldrich C0832 Critical Commercial Assays SuperScript III kit Invitrogen 18080-051 Accuprime Taq polymerase Invitrogen 12339-024 AmpliTaq Gold 360 Master Mix Invitrogen 4398886 Absolutely RNA Nanoprep kit Agilent 400753 RNAscope Manual Fluorescent Multiplex kit V2 Advanced Cell Diagnostics 323100 Deposited Data TRAP RNA-Seq raw and analyzed data This paper GEO: GSE124681 Experimental Models: Organisms/Strains Mouse, B6.129(Cg)-Fostm1.1(cre/ERT2)Luo/J The Jackson Laboratory 021882 Mouse, B6;129S4-Gt(ROSA)26Sortm9(EGFP/Rpl10a)Amc/J The Jackson Laboratory 024750 Mouse, ChatCre The Jackson Laboratory 006410 Mouse, Slc17a6Cre The Jackson Laboratory 016963 Mouse, CckCre The Jackson Laboratory 012706 Mouse, GalCre GENSAT KI87; RRID: MMRRC_ 031060-UCD Mouse, CalcaCre Dr. Richard D. Palmiter NA (Continued on next page) e1 Cell 177, 1–15.e1–e6, May 16, 2019

Techniques: Labeling, Activation Assay, Injection