1. Volume 10, Issue 2, Pages 386-398 (August 2004)
A Facile Nonviral Method for Delivering Genes and siRNAs to Skeletal Muscle of Mammalian Limbs 
James E. Hagstrom, Julia Hegge, Guofeng Zhang, Mark Noble, Vladimir Budker, David L. Lewis, Hans Herweijer, Jon A. Wolff 
Molecular Therapy 
Volume 10, Issue 2, Pages (August 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
(A, left) Schematic diagram of catheter-mediated intravenous injection of nucleic acids into distal hind limb of rats. (A, right) Venous vasculature of rat limb and sites of pDNA injection (as indicated by arrows). (B–D) Effects of volume of injection (B), rate of injection (C), and dose of pDNA delivered (D) on luciferase expression following intravenous delivery of pDNA (pCI-Luc-K) into the hind limbs of female Sprague–Dawley rats (120–150 g). To determine reporter gene expression levels, all muscle groups of the lower limb (6 groups) were harvested and weighed, cell lysates were prepared, and luciferase assays were performed on the lysates of each muscle group. Luciferase expression is indicated as an average of all groups in nanograms luciferase per gram of muscle tissue. For each data point, two to seven limbs were injected and analyzed. T bars indicate standard deviation.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Photomicrographs of rat limb muscles stained for β-galactosidase following single (left) or repeat (triple) intravenous injections (right) of 500 μg of pDNA (pCI-LacZ). Corresponding relative values for β-galactosidase expression from (A) gastrocnemius and (B) shin muscle groups are displayed just above the β-galactosidase-stained images as determined by a chemiluminescence assay. (A) Images were captured at 100× magnification and (B) images were captured at 200× magnification.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Intravascular injection of therapeutic genes into mammalian limbs. (A) Time course of erythropoietin expression following injection of 500 μg pDNA (in 3 ml NSS/20 s) encoding rat erythropoietin into the great saphenous vein of the distal limb of 120- to 150-g female Sprague–Dawley rats (Harlan Laboratories). Injections (n = 3) were performed as described for Fig. 1. (B) Immunohistochemical staining for human dystrophin expression in mdx4cv mouse muscle (from gastrocnemius) 1 week after intravenous injection of 300 μg of a pDNA human dystrophin expression vector [46] in 0.6 ml of NSS (7.5 s injection). (C) Similar staining procedure but mdx4cv mice were injected with pCI-Luc to serve as a negative control. The primary antibody used for detection of the human dystrophin in these studies was a monoclonal NCL-DYS3 (Novocastra Laboratories). (B and C) Captured at 160× total magnification.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Schematic diagram of catheter-mediated intravenous injection of nucleic acids into distal hind limb of rhesus macaques (A, left). (A, right) Venous vasculature of rhesus limb with the site of pDNA (and siRNA) injection (pCI-Luc-K and pCI-LacZ) indicated by small arrow. (B, C, D) Photomicrographs from three different lower limb muscle groups stained for β-galactosidase (at 7 days postinjection) following a single intravenous injection of 40 mg of pDNA (pCI-LacZ) into a distal site of the great saphenous vein (primate 3, see Table 3 for methods). Individual images indicate representative high-expressing areas in two different locations of the named muscle group. Percentages of β-galactosidase-positive cells are indicated above each image. The muscle descriptions indicate the following muscle groups of the hindlimb: gastrocnemius, posterior muscle group of lower leg; soleus, medial muscle group of lower leg; extensor hallucis brevis, muscle group of the dorsal foot. (B) Captured at 100× total magnification and (C and D) captured at 200×.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
RNA interference in rat and primate limb muscle following intravenous codelivery of siRNAs and pDNA expression vectors. (A) In rat plasmid DNAs encoding firefly luciferase (pSP-luc+) and pansy luciferase (pRL-SV40) were injected into the great saphenous vein along with either control siRNAs (EGFP; n = 5) or siRNAs targeted against the firefly luciferase (n = 5). Firefly luciferase knockdown in limb muscle using the targeted siRNA was plotted against pansy luciferase knockdown using the control siRNA (EGFP), which was normalized to 1. For rat injections, T bars represent standard deviation from animal to animal. (B) In primate, plasmid DNAs encoding firefly luciferase (pCI-Luc-K) and pansy luciferase (pCI-Renilla) were injected into alternate front and hind limbs via the cephalic vein (front limb) and the great saphenous vein (hind limb). Along with pDNAs, control siRNAs (EGFP) were co-injected into the hind limb, and siRNAs targeted against the firefly luciferase were co-injected into the front limb. Four days postinjection, the animal was euthanized and individual muscle groups were harvested, and firefly luciferase expression using the targeted siRNA (mean luciferase expression in front limb muscle groups) was plotted against firefly luciferase knockdown using the control siRNA (mean luciferase expression in hind-limb muscle groups), which was normalized to 1. For primate studies, T bars represent standard deviation in target gene expression levels in different muscle groups of the limb from one animal (n = 20 muscle groups).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Time course of creatine phosphokinase (CPK) values following two sequential intravenous injections in (A) beagle and (B) rhesus macaques. In both the dog and the monkeys, injections were performed on days 0 and 3 (indicated by arrows). In the dog, arrows indicate that an intravenous injection was done into one foreleg on day 0 and into the contralateral foreleg on day 3. In the monkeys, arrows indicate that intravenous injections were done in each animal (into one arm and leg) on day 0 and the contralateral arm and leg on day 3 (i.e., two injections at day 0 and two injections at day 3). The day 3 blood sample (for CPK analysis) in both species was collected prior to the second intravenous injection. All animals were sacrificed at 7 days postinjections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions