1. Volume 16, Issue 5, Pages 907-915 (May 2008)
A Receptor-targeted Nanocomplex Vector System Optimized for Respiratory Gene Transfer 
Aristides D Tagalakis, Robin J McAnulty, James Devaney, Stephen E Bottoms, John B Wong, Martin Elbs, Michele J Writer, Helen C Hailes, Alethea B Tabor, Christopher O'Callaghan, Adam Jaffe, Stephen L Hart 
Molecular Therapy 
Volume 16, Issue 5, Pages (May 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Structures of lipids and peptides. DHDTMA/DOPE is the lipid component of LED-1. DHDTMA has an alkyl tail with 16 carbons and a cis bond at position 11. DOSEP3 is a cationic lipid with an 18-carbon alkyl chain and a polyethylene glycol linker. The peptide has the generic format K16GACSERSMNFCG where K16 mediates DNA binding; the residues in bold comprise a receptor-binding ligand flanked by cysteine residues to constrain the peptide structure by formation of a disulphide bridge, and the glycyl-l-alanine (GA) dipeptide is a spacer. The peptide mediates DNA compaction and the exposed ligand targets the complex to cell surface receptors.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
In vitro transfections reveal that LED-1 is more efficient at transfecting four different epithelial cell lines. (a) Transfections with LED-1, GL67, polyethylenimine (PEI) 22 kd, and PEI 25 kd complexed with pCILux were performed in two normal (16HBEo– and 1HAEo–) and two cystic fibrosis (CFBE41o– and CFTE29o–) human airway epithelial cell lines. The N/P ratio for both PEI 22 kd and PEI 25 kd was 10:1. Inall transfections 0.25 μg of DNA/well was used. Twenty-four hours after transfection a luciferase assay was performed followed by a Bradford protein assay. The experiment was repeated on two occasions. Each result is the mean of six values and error bars represent the SD about the mean (mean ± SD). The graph was plotted using a logarithmic scale for the, y-axis. (b) Transfections with LED-1, GL67, PEI 22 kd, and PEI 25 kd complexed with pEGFP-N1 were also performed in these cell lines. The N/P ratio for both PEI 22 kd and PEI 25 kd was 10:1. In all transfections 0.25 μg of DNA/well was used. Twenty-four hours after transfection wells were photographed and then fixed and assayed for green fluorescent protein expression by flow cytometry. Each result is the mean of three values and error bars represent the SD about the mean (mean ± SD).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Comparison of in vivo transfection efficacy of several nonviral vectors after a single or triple dose regime. Luciferase activity in whole-lung lysates 24 hours following instillation after 1 or 3 weekly administrations of four different vectors. Luciferase activity is significantly reduced following the third instillation for both polyethylenimine (PEI) 22 kd (P < 0.01) and GL67 (P < 0.05) but not with the LED-1 or the LED-2 vector. The graph was plotted using a logarithmic scale for the y-axis. RLU, relative light unit.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Immunohistochemical localization of luciferase following transfection with various vectors. Representative sections are shown from animals transfected with LED-1 (a–e), LED-2 (f–j), GL67 (k–o) and polyethylenimine (PEI) 22 kd (p–t) complexed with pCI (a,f,k,p) or pCILux (b,c,g,h,l,m,q,r) following a single instillation or following 3 weekly administrations (d,e,i,j,n,o,s,t) at low (b,d,g,i,l,n,q,s) and high (a,c,e,f,h,j,k,m,o,p,r,t) magnification. Sections from animals transfected with pCI acted as negative controls and showed no positive staining. Transfection following a single instillation of vectors with pCILux showed brown staining, indicative of luciferase expression, which was predominantly localized to the bronchial epithelium (denoted by *low, and Ep in high, magnification images) with LED-1 (b,c) and PEI 22 kd (q,r). In contrast, for LED-2 (h) and GL67 (m) staining was predominantly associated with macrophages (Mac). Following 3 weekly instillations, with LED-1 (d,e) and PEI 22 kd (s,t) staining was still predominantly associated with the bronchial epithelial. In contrast to the single instillation, moderate staining of the bronchial epithelium was apparent following repeated transfection with LED-2 (i,j) and GL67 (n,o). Four or five animals were analyzed per group and staining was consistent between animals in each group. The bar scale of 50 μm applies to all high power images, whereas the bar scale of 400 μm applies to all low power images. Ep, epithelial cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Inflammatory mediator profile in bronchoalveolar lavage fluid following intratracheal instillation of three different vector formulations (with pCILux or pCI). All vectors induced an inflammatory response with both plasmids pCI and pCILux, with all inflammatory cytokines elevated. In all cases interleukin-12 (IL-12) was elevated with the exception of polyethylenimine (PEI) 22 kd, which showed though the highest levels for cytokines IL-1b, IL-2, IL-4, IL-5, and IL-6. Each value represents the mean ± SEM for at least six animals and is expressed as picogram per 100 μl.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Effect of vector instillation on lung cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. In untreated control lung (a) only occasional apoptotic bronchial epithelial cells (Ep) were observed (stained black). Instillation of GL67 complexed with pCI (b) induced an increase in macrophage (Mac) numbers, of which many appeared to be apoptotic. In addition, areas of alveolar epithelial cell (*) apoptosis were also observed. Transfection with LED-1 complexed with pCI (c) also showed a small increase in bronchial epithelial cell apoptosis. Instillation of polyethylenimine (PEI) 22 kd complexed with pCI (d) showed a similar but much more extensive bronchial and alveolar epithelial cell apoptosis to that observed with LED-1. The bar scale of 50 μm applies to all images.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Reverse transcription-PCR (RT-PCR) analysis of expression of human cystic fibrosis transmembrane conductance regulator (CFTR) gene following in vivo intratracheal instillation of LED-1/pcDNA3.CFTR. Lanes 1–5 are lungs from CFTR-transfected mice and lane 6 are lungs from an untransfected mouse. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control of the suitability of the RNA used in the RT-PCR.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

9. Figure 8
Compatibility of the receptor-targeted nanocomplex with nebulization. LED-1 vector was nebulized using the Aerogen Pro nebulizer and the collected nebulized material was then instilled intratracheally into mice. Mice were instilled intratracheally with 50 μl of nebulized LED-1 vector containing 1.5 μg of DNA. The nebulized material was biologically active as it transfected the lungs with efficiencies somewhere between the 8 and the 16 μg of pCILux, which were instilled without prior nebulization. RLU, relative light unit.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions