A BMP/Activin A Chimera Induces Posterolateral Spine Fusion in Nonhuman Primates at Lower Concentrations Than BMP-2.

BACKGROUND: Supraphysiologic bone morphogenetic protein (BMP)-2 concentrations are required to induce spinal fusion. In this study, a BMP-2/BMP-6/activin A chimera (BV-265), optimized for BMP receptor binding, delivered in a recombinant human collagen:CDHA [calcium-deficient hydroxyapatite] porous composite matrix (CM) or bovine collagen:CDHA granule porous composite matrix (PCM), engineered for optimal BV-265 retention and guided tissue repair, was compared with BMP-2 delivered in a bovine absorbable collagen sponge (ACS) wrapped around a MASTERGRAFT Matrix (MM) ceramic-collagen rod (ACS:MM) in a nonhuman primate noninstrumented posterolateral fusion (PLF) model. METHODS: In vivo retention of 125I-labeled-BV-265/CM or PCM was compared with 125I-labeled-BMP-2/ACS or BMP-2/buffer in a rat muscle pouch model using scintigraphy. Noninstrumented PLF was performed by implanting CM, BV-265/CM, BV-265/PCM, or BMP-2/ACS:MM across L3-L4 and L5-L6 or L3-L4-L5 decorticated transverse processes in 26 monkeys. Computed tomography (CT) images were acquired at 0, 4, 8, 12, and 24 weeks after surgery, where applicable. Manual palpation, µCT (microcomputed tomography) or nCT (nanocomputed tomography), and histological analysis were performed following euthanasia. RESULTS: Retention of 125I-labeled-BV-265/CM was greater than BV-265/PCM, followed by BMP-2/ACS and BMP-2/buffer. The CM, 0.43 mg/cm3 BMP-2/ACS:MM, and 0.05 mg/cm3 BV-265/CM failed to generate PLFs. The 0.15-mg/cm3 BV-265/CM or 0.075-mg/cm3 BV-265/PCM combinations were partially effective. The 0.25-mg/cm3 BV-265/CM and 0.15 and 0.3-mg/cm3 BV-265/PCM combinations generated successful 2-level PLFs at 12 and 24 weeks. CONCLUSIONS: BV-265/CM or PCM can induce fusion in a challenging nonhuman primate noninstrumented PLF model at substantially lower concentrations than BMP-2/ACS:MM. CLINICAL RELEVANCE: BV-265/CM and PCM represent potential alternatives to induce PLF in humans at substantially lower concentrations than BMP-2/ACS:MM.

West Nile Virus Seroprevalence in an Outdoor Nonhuman Primate Breeding Colony in South Florida.

West Nile virus (WNV) was first detected in Florida in July 2001, with 404 human cases reported to the Centers for Disease Control and Prevention as of February 2020. The subtropical climate of Florida is ideal for the mosquitoes that transmit WNV. We investigated the WNV seroprevalence in 3 NHP species housed outdoors at The Mannheimer Foundation in South Florida. From January to December 2016, 520 3 to 30 y old NHP were sampled at our 2 closed sites in Homestead and LaBelle: 200 rhesus macaques (Macaca mulatta), 212 cynomolgus macaques (Macaca fascicularis), and 108 hamadryas baboons (Papio hamadryas hamadryas). The presence of WNV IgG antibodies in these animals was determined by serum neutralization assays, which found a total seroprevalence of 14%. Seroprevalence was significantly higher in the baboons (29%) than the rhesus (11%) and cynomolgus (9%) macaques. The probability of seropositivity significantly increased with age, but sex and site did not significantly affect seroprevalence. The frequency of WNV seropositivity detected in these outdoor-housed NHP suggests that screening for WNV and other vector-borne diseases may be necessary prior to experimental use, particularly for infectious disease studies in which viremia or viral antibodies could confound results, and especially for populations housed outdoors in warm, wet climates. As no seropositive subjects demonstrated clinical signs of WNV and WNV exposure did not appear to significantly impact colony health, routine testing is likely unnecessary for most NHP colonies. However, WNV infection should still be considered as a differential diagnosis for any NHP presenting with nonspecific neurologic signs. Mosquito abatement plans and vigilant sanitation practices to further decrease mosquito and avian interaction with research NHP should also be considered.

A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix.

Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.

Basics of macaque pediatrics.

It is clear that raising infants with their mothers and family group is ideal for their appropriate development. When this is not feasible, it is necessary to try to detect abnormalities beforehand and avoid circumstances that can be detrimental to the animal's social and reproductive behavior. It is of great importance to try to simulate as much as possible an environment adequate enough to develop a species-specific behavior. Multiple measurements and parameters exist to evaluate macaque infants. Although infants are vulnerable to multiple health problems, with effort and dedication and 24-hour care, many times these can be corrected.

Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.

Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.

Spontaneous primary squamous cell carcinoma of the lung in a rhesus macaque (Macaca mulatta).

A 3-y-old male rhesus macaque (Macaca mulatta) was noticed to be lethargic in the compound. Physical exam revealed cyanotic mucous membranes, dyspnea, bilateral harsh lung sounds, wheezing on expiration, and a firm mass possibly associated with the liver. Radiographs revealed bilateral soft tissue opacities in the thorax. Due to poor prognosis, the rhesus was euthanized, and a necropsy was performed. Both right and left lung lobes were consolidated and had multifocal white-tan masses. On cut section, the masses were firm, had areas of necrosis, hemorrhage, and often contained a tenacious exudate. Masses were identified in the liver and both kidneys. Given the morphologic features of the neoplasm, a diagnosis of squamous cell carcinoma was made. Immunohistochemistry staining for thyroid transcription factor, a nuclear transcription factor normally found in lung, thyroid, and tumors arising from either of those tissues, confirmed that the masses originated from the lung. Malignant primary lung tumors are divided into 8 main histologic subtypes: squamous cell carcinoma, small-cell carcinoma, large-cell carcinoma, adenocarcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, carcinoid tumor, and salivary gland tumors. Clinical signs associated with lung tumors include, but are not limited to, dyspnea, coughing, hemoptysis, lethargy, anorexia, and weight loss. Although squamous cell carcinoma will be low on the differential list for these clinical signs, we encourage clinicians and researchers to not rule it out solely based on incidence and age of the animal.

Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.

Spinal muscular atrophy (SMA), the most common autosomal recessive neurodegenerative disease affecting children, results in impaired motor neuron function. Despite knowledge of the pathogenic role of decreased survival motor neuron (SMN) protein levels, efforts to increase SMN have not resulted in a treatment for patients. We recently demonstrated that self-complementary adeno-associated virus 9 (scAAV9) can infect approximately 60% of motor neurons when injected intravenously into neonatal mice. Here we use scAAV9-mediated postnatal day 1 vascular gene delivery to replace SMN in SMA pups and rescue motor function, neuromuscular physiology and life span. Treatment on postnatal day 5 results in partial correction, whereas postnatal day 10 treatment has little effect, suggesting a developmental period in which scAAV9 therapy has maximal benefit. Notably, we also show extensive scAAV9-mediated motor neuron transduction after injection into a newborn cynomolgus macaque. This demonstration that scAAV9 traverses the blood-brain barrier in a nonhuman primate emphasizes the clinical potential of scAAV9 gene therapy for SMA.

Safingol toxicology after oral administration to TRAMP mice: demonstration of safingol uptake and metabolism by N-acylation and N-methylation.

Safingol [(2S,3S)-2-amino-1,3-octadecanediol] is an unnatural l-threo-stereoisomer of sphinganine that is cytotoxic for cancer cells in culture and is being tested in phase 1 human clinical trials. To determine if safingol can be absorbed orally and if it affects prostate cancer in a mouse strain used in prostate cancer studies, safingol was fed to TRAMP (transgenic adenocarcinoma of mouse prostate) mice for 2 weeks at 0.0125% to 0.1% w/w of the diet. Analysis of safingol and safingol metabolites in blood and tissues by liquid chromatography electrospray ionization tandem mass spectrometry revealed uptake in tissue and extensive conversion of safingol to N-acyl species (comparable to natural "ceramides") and mono-, di-, and tri-N-methyl metabolites that have not been observed previously. Safingol caused significant hepatotoxicity at all dosages, as reflected in elevated liver alanine aminotransferase, and at the highest dose (0.1 %) caused changes in liver histology (appearance of autophagosomal vacuoles) and renal toxicity (based on elevation of blood urea nitrogen) and decreases in packed blood cell volume and body weight. Safingol did not inhibit the prostate pre-neoplastic lesion (prostate intraepithelial neoplasia) in TRAMP mice; however, additional studies at lower dosages for longer time were not pursued due to host toxicity. Safingol and its N-methyl metabolites were cytotoxic to both a human prostate cell line (DU145) and mouse BALB 3T3 cells; therefore, the host and potential antitumor toxicity may be due to multiple molecular species of safingol.

Evaluation and comparative analysis of a technique for laparoscopic ovariectomy in rhesus macaques (Macaca mulatta).

Surgical ovariectomy is commonly performed to support studies involving the rhesus macaque (Macaca mulatta). Traditionally, these procedures have been performed via laparotomy. We developed a laparoscopic approach to reduce surgical pain, decrease convalescence times, and reduce the total number of animals necessary to obtain valuable scientific data in studies requiring repeated intra-abdominal access in conjunction with ovariectomy. We used our new technique to perform laparoscopic ovariectomy on 8 adult female rhesus macaques; 2 additional animals underwent ovariectomy via laparotomy; data for these prospective groups were compared with retrospective data from conventionally ovariectomized macaques. The surgical time (ST; mean +/- standard error) for the laparoscopic procedures was 68 +/- 3 min, with a return-to-group time (RTG) of 8 +/- 1 d. In comparison, ST for the retrospective group was 54 +/- 3 min, with a mean RTG of 33 +/- 5 d (range, 15 to 60 d). ST differed significantly between groups. ST for the laparoscopic procedure was longer (mean difference, 14 min; 95% confidence interval, 6 to 21 min), but laparotomized animals consistently had higher RTG (6 d for the prospective group [n = 2] and 25 d for the retrospective group [n = 9]). All study animals had estradiol levels of less than 5.6 pg/ml at 6 mo after surgery. We therefore conclude that laparoscopic ovariectomy of rhesus macaques is a safe and effective technique that may reduce postoperative recovery times.

Spontaneous cutaneous squamous cell carcinoma in a sooty mangabey (Cercocebus atys): a case report.

An adult sooty mangabey (Cercocebus atys) with a solid mass arising from the skin of the dorsolateral cervical area was presented to the veterinary clinical staff. Grossly, the mass was firm, elongated, ulcerated at the tip, and measured 2.7 x 2.0 x 2.3 cm. It was surgically excised and then submitted for histopathologic evaluation. On histopathology, this tumor was composed of irregular masses and cords of neoplastic squamous epithelial cells that invaded the dermis and subcutis, often undergoing keratinization and forming numerous keratin pearls. On the basis of these histologic findings, the mass was diagnosed as a squamous cell carcinoma. Additional tests, including hematologic evaluations and radiographic views of the abdominal, thoracic, and cervical areas, were normal. Sections of the tumor were analyzed by electron microscopy and showed no evidence of viral particles. To the authors' knowledge, this is the first reported case of a spontaneous cutaneous squamous cell carcinoma in a sooty mangabey.