Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer.

ABSTRACT: Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e-4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

The Use of Waterless Alcohol-based Antiseptic for Surgical Skin Preparation in Rhesus Macaques (Macaca mulatta).

Ensuring asepsis of the surgical site before surgery is an essential component of safe surgical practices to reduce the incidence of surgical site infections in veterinary medicine. The current accepted method of skin preparation is a multistep process that alternates either a povidone-iodine or chlorhexidine soap scrub with a 70% alcohol rinse. After cleansing, the site is left to dry before draping. The goal of this study was to assess the effectiveness of a waterless alcohol-based (WAB) antiseptic as part of a 2-step procedure after the soap scrub. WAB antiseptics are commonly used as a presurgical hand scrub for the surgeon as they evaporate quickly and provide effective antisepsis. Previous studies have examined the WAB antiseptics in small animal surgeries. We tested this approach in large animal surgery. Twenty-four rhesus macaques were divided into 4 groups that received one of the following treatments: saline and alcohol, iodine-alcohol-iodine, soap scrub/WAB, and chlorhexidine-alcohol-chlorhexidine. The surgical site was swabbed before and after treatment and plated to assess sterility. Overall, no colonies were recovered from skin treated with WAB antiseptic, establishing it as an effective alternative to the current standard protocol. This method will simplify the current 3-step procedure and reduce animal handling, the use of materials, and the time necessary for surgical preparation.

Low profile halo head fixation in non-human primates.

BACKGROUND: We present a new halo technique for head fixation of non-human primates during electrophysiological recording experiments. Our aim was to build on previous halo designs in order to create a simple low profile system that provided long-term stability. NEW METHOD: Our design incorporates sharp skull pins that are directly threaded through a low set halo frame and are seated into implanted titanium foot plates on the skull. The inwardly directed skull pins provide an easily calibrated force against the skull. RESULTS: This device allowed for head fixation within 1 week after implantation surgery. The low-profile design maximized the area of the skull available and potential implant orientations for electrophysiological experiments. It was easily maintained and was stable in 2 animals for the 6-8 months of testing. The quality of single unit neural recordings collected while using this device to head fix was indistinguishable from traditional head-post fixation. The foot plates used in this system did not result in significant MRI distortion in the location of deep brain targets (∼0.5mm) of a 3D printed phantom skull. COMPARISON WITH EXISTING METHOD(S): The low profile design of this halo design allows greater access to the majority of the frontal, parietal, and occipital skull. It has fewer parts and can hold larger animals than previous halo designs. CONCLUSIONS: Given the stability, simplicity, immediate usability, and low profile of our head fixation device, we propose that it is a practical and useful means for performing electrophysiological recording experiments on non-human primates.

Efficacy of Intravenous Mannitol in the Management of Orbital Compartment Syndrome: A Nonhuman Primate Model.

PURPOSE: To report the efficacy of intravenous mannitol in the treatment of orbital compartment syndrome. METHODS: An experimental study was conducted on 4 nonhuman primates (8 orbits). Orbital compartment syndrome was simulated by injecting autologous blood into both orbits of each nonhuman primate until a pressure of 80 mm Hg was reached (time 0). After 10 minutes, nonhuman primates were randomized to receive an infusion of either mannitol or saline, given over 15 minutes. Five minutes after the infusion was complete, lateral canthotomy and cantholysis was performed on both orbits in isolated steps every 5 minutes. During the study protocol, orbital and intraocular pressures were recorded every 5 minutes, with a final set of measurements at 60 minutes. The primary outcome measures were the mean change in pressure from time 0 to 60 minutes, as well as the mean change in pressure during the infusion period. RESULTS: There was no statistically significant difference in the mean changes in orbital or intraocular pressure from time 0 to 60 minutes of the protocol. However, during the infusion period there was significantly greater decrease in both orbital and intraocular pressure in the mannitol compared with saline group (-34.0 vs. -9.3 mm Hg for orbital pressure [p = 0.03]; -34.8 vs. -9.7 mm Hg for intraocular pressure [p = 0.04]). CONCLUSIONS: While the definitive treatment of orbital compartment syndrome is lateral canthotomy and cantholysis, mannitol results in a rapid and clinically meaningful drop in orbital and intraocular pressure. The authors believe that their data support the routine use of mannitol in orbital compartment syndrome, especially when there is a delay in timely surgical management.

Hemodynamic and behavioral differences after administration of meloxicam, buprenorphine, or tramadol as analgesics for telemeter implantation in mice.

Cannulation of the common carotid artery for chronic, continuous radiotelemetric recording of aortic hemodynamic properties in mice is a highly invasive recovery surgery. Radiotelemetric recording, by its continuous nature, gives the most accurate measurements of hemodynamic variables in experimental animals, and is widely used in the study of cardiovascular diseases including hypertension. The American Heart Association has recommended data acquisition by radiotelemetric recording but did not provide guidelines regarding postoperative analgesic support. We assessed hemodynamic parameters, locomotor activity, food intake, and weight loss in radiotransmitter-implanted CD1 female mice receiving analgesic support during the first 48 h after surgery. The efficacy of analgesic support from the NSAID meloxicam was compared with that of the widely used opioid agonist buprenorphine and the related compound, tramadol. Meloxicam-treated mice recovered lost body weight more rapidly than did tramadol-or buprenorphine-treated mice. Furthermore, meloxicam-treated mice maintained circadian rhythm after surgery and had tighter regulation of mean arterial pressure than did tramadol- or buprenorphine-treated mice. Meloxicam was also superior with regard to food intake, locomotor activity, and limiting variance in hemodynamic parameters. This study indicates that when compared with buprenorphine and tramadol, meloxicam should be the postoperative analgesic of choice for radiotelemeter implantation in mice.

Comparison of buprenorphine and meloxicam for postsurgical analgesia in rats: effects on body weight, locomotor activity, and hemodynamic parameters.

Buprenorphine is administered to humans and animals for postoperative pain management, although its use is associated with complications. Alternative analgesics, including the nonsteroidal antiinflammatory meloxicam, are available, but information on their postoperative effects is limited. The objective of the present study was to compare buprenorphine (0.03 mg/kg SC twice daily for 3 d) with meloxicam (2 mg/kg SC initial dose followed by 1 mg/kg SC once daily for 2 d) by assessing parameters relating to postsurgical recovery in rats that underwent surgical implantation of radiotelemetric transducers. Rats treated after surgery with buprenorphine showed greater reductions in body weight, food consumption, locomotor activity, and nighttime heart rates than did meloxicam-treated rats. Buprenorphine and meloxicam treatments both had stimulatory effects on mean arterial pressure and daytime heart rate measurements, although effects on nighttime mean arterial pressure were greater in the buprenorphine-treated rats. In summary, the lesser physiologic changes associated with meloxicam, as compared with buprenorphine, suggest that meloxicam offers advantages for use as a postoperative analgesic after laparotomy and radiotelemetric transducer implantation in rats.