A heterologous intron incorporated into a TG cassette of a lentiviral vector results in increased transgene expression, measured as an increase in NGF output. A) Maps of the lentiviruses carrying TG expression vector constructs. B) NGF release into the culture medium of 293T cells transduced with the viral vectors shown in A. Bars = mean ± SEM; numbers above bars are numbers of wells per group; *P < 0.05 (t-test) vs. no intron group. NGF was measured after 24 h of culture. See figure legend 1 and text for descriptions and definitions of vector components and abbreviations.

Maps of the 3^rd generation lentiviral vector system containing either a TG or RNAi (TG-RNAi) cassette. The 5’LTR contains the heterologous U3 promoter (htU3), the repeat region/transcription initiation site (R), and the polyadenylation region (U5). The R and U5 regions are also present in the 3’LTR but importantly there is a 400 bp deletion in the 3’LTR U3 region (ΔU3) that results in the self-inactivation (SIN) of these vectors. The heterologous promoters are either the Rous sarcoma virus promoter (RSVp) or the cytomegalovirus promoter (CMVp); the CMVp also serves as the TG-RNAi cassette promoter. The other components include the packaging signal (ψ), the Rev response element binding site (RRE), the central polypurine tract (cPPT), and the woodchuck-hepatitis-virus posttranslational regulatory element (wPRE). See section 2.3 for descriptions of the vector components. A) Map of a lentivirus delivery vector [32] containing a bicistronic TG cassette composed of a human nerve growth factor (hNGF) cDNA linked to an enhanced green fluorescent protein (eGFP) cDNA via an internal ribosome entry site (IRES) and placed downstream of the rat insulin II intron A (Int). B) Map of the pPRIME lentivirus delivery vector [37] containing an artificial miR30 and replaceable hairpin structure able to accommodate 1 to 3 hairpins [42]; Xho I and Eco RI restriction enzyme sites are used as cloning sites to insert the hairpin(s). C) Maps of the packaging system containing the plasmids gB gag LV [50], pLP2, and pLP/VSVG (Invitrogen). The mutation noted in the gag gene increases the ability of the HIV-1 based lentiviral vector to transduce simian cells [31]. The other components in the packaging plasmids are the β-globin polyadenylation signal sequence (β-globin pA), HIV-1 polyadenylation signal sequence (HIV-1 pA), and the vesicular stomatitis virus glycoprotein (VSV-G). See section 2.4 for more information regarding the packaging plasmid components.

X-ray and MRI guided stereotaxic injection of lentiviral particles into the arcuate nucleus of the hypothalamus in rhesus macaques. A) Initial x-ray showing the clinoids of the sella turcica (arrowheads). B) Drawing of a monkey skull showing the placement of fiducial markers. The most posterior marker (arrow) was placed on the midline and used to establish the true midline of the brain. C) MRI in which the posterior fiducial marker can be seen (arrow). D) MRI showing the arcuate nucleus of the hypothalamus (arrow). E) Final x-ray showing the injection needle (arrow); the tip of the needle shows the injection site, which is 1 mm dorsal to the line drawn between the clinoids (arrowhead). F) MRI following bilateral injection of 1.5 µ1 of a 1 µM solution of MnCl in 0.4% trypan blue (in phosphate buffered saline) into the arcuate nucleus of the hypothalamus (arrow). G) Gross section of a monkey brain following bilateral injection of MnCl-trypan blue showing the sites of the injections (arrows).