Herpesvirus-Mediated Systemic Delivery of Nerve Growth Factor Darren Wolfe, William F. Goins, Theodore J. Kaplan, Saverio V. Capuano, Julie Fradette, Michael Murphey-Corb, Paul D. Robbins, Justus B. Cohen, Joseph C. Glorioso  Molecular Therapy  Volume 3, Issue 1, Pages 61-69 (January 2001) DOI: 10.1006/mthe.2000.0225 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Detection of NGF in synovial fluid following intra-articular delivery of the HCMV-NGF vector SHN. Pairs of rabbits were infected with the 108 pfu of the NGF-expressing vector SHN and knee lavage levels of NGF were quantitated by ELISA as described under Material and Methods. Bars represent the range of NGF levels observed within each animal group. Total amounts of NGF per knee are plotted over time. Molecular Therapy 2001 3, 61-69DOI: (10.1006/mthe.2000.0225) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Long-term lacZ expression following delivery of SHZ into the knee joint of rabbits. (A) X-gal staining of the ACL 90 days following intra-articular delivery of 1 × 108 pfu. (B) PCR analysis of DNA isolated from the ACL 90 days after intra-articular delivery comparing lacZ-positive and lacZ-negative regions of the ACL. (C) X-gal staining of the patellar ligament 90 days following direct patellar ligament inoculation of 2 × 107 pfu of SHZ. (D) PCR analysis of DNA isolated from patellar and anterior cruciate ligaments 90 days after direct patellar ligament injection of SHZ or d120. Pos, positive control; Neg, negative control; LacZ+, DNA from a lacZ-positive ACL from an SHZ-injected knee; LacZ−, DNA from a lacZ-negative ACL; PL1, 2, patellar ligament DNA from a d120-injected knee; AL1, 2, ACL DNA from the same knee; PR1, 2, patellar ligament DNA from an SHZ-injected knee; AR1, 2, ACL DNA from the same knee. Molecular Therapy 2001 3, 61-69DOI: (10.1006/mthe.2000.0225) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Sustained systemic levels of NGF following delivery of NGF-expressing vectors SHN (A, C) or SLN (B, C) by intra-articular (A, B) or patellar ligament (C) injection. Groups of two (intra-articular) or three (patellar) rabbits were injected with 1 × 108 pfu vector (SHN, SLN, or SHZ) and plasma levels of NGF were quantitated by ELISA. The average NGF level is plotted with bars representing the range of NGF for each experimental group. RT-PCR with primers specific for vector-derived NGF sequences (D) revealed vector-derived transcripts driven by either HCMV (H) or LAP2 (L) present in cells of infected patellar ligaments (I) but not the contralateral ligaments (C). Molecular Therapy 2001 3, 61-69DOI: (10.1006/mthe.2000.0225) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Sustained expression and bioactivity of NGF upon SHN delivery into the joint space of nonhuman primates. (A) Plasma levels of NGF in a rhesus macaque injected intra-articularly with 108 pfu SHN and a second macaque injected with 108 pfu of the control vector SHZ, as determined by ELISA. (B) NGF bioassay on rhesus serum 6 months post-vector injection. PC12E2 cells were incubated for 4 days in the presence of 20% serum samples from the control (SHZ-injected) or NGF-producing (SHN-injected) macaque. Molecular Therapy 2001 3, 61-69DOI: (10.1006/mthe.2000.0225) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Epidermal neurites visualized by immunohistochemistry from footpad biopsy from SHN-injected rhesus macaques 6 months post-intra-articular vector delivery. Image is phase-contrast microscopy with immersion oil at 100×. Epidermal neurites are evident projecting into the epidermis from the dermal layer of the skin. Molecular Therapy 2001 3, 61-69DOI: (10.1006/mthe.2000.0225) Copyright © 2001 American Society for Gene Therapy Terms and Conditions