Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at approximate to 0.4 gestation. The initial cohort received 1 x 10(12) vector genomes (vgs) of AAV5-hFIX (n = 5; 0.45 x 10(13) vg/kg birth weight), resulting in approximate to 3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 x 10(13) vg boluses. AAV5-hFX animals (n = 3; 3.57 x 10(13) vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients (n = 3; 2.56 x 10(13) vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX (n = 6, 3.12 x 10(13) vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 x 10(11) vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC invivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implantedxFVIII activity secreted/million BMSCs/24hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.

The success of an animal care and use program depends on various factors, including a work environment in which laboratory animal care and research personnel can perform their work without risking their health and safety. Personnel are exposed to various hazards in performing veterinary care, husbandry care, and research experiments. Therefore, it is imperative that institutions maintain a robust and comprehensive occupational health and safety program (OHSP) that can effectively assess and mitigate risks and address exposures. Examples of hazards include chemicals used for sanitation (for example, disinfectants and descaling acids), inhalant anesthetics, compounds or toxins used to create animal models, some therapeutic or experimental drugs and interventions (for example, carcinogenic or chemotherapeutic agents), infectious organisms and potentially infected materials, radiation, ergonomic hazards, noise, and allergens. According to the Guide for the Care and Use of Animals, the OHSP must be consistent with federal, state, and local regulations.9 The federal law Occupational Safety and Health (OSH) Act (29 CFR 15) applies to most private sector employers and their workers and some public sector employers and their workers in the U.S. Compliance with regulations and standards under this law can be enforced either directly through the OSH Administration (OSHA) or through an OSHA-approved state plan.13 Other documents also contribute to the OHSP regulatory framework of an animal care and use program. Foremost of these is the Occupational Health and Safety in the Care and Use of Research Animals, created by the Committee on Occupational Safety and Health in Research Animal Facilities, Institute of Laboratory Animal Resources.11 Published in 1997, this remains to be the authoritative guidance on the occupational health and safety of personnel in the animal care and use program. Other pertinent regulatory documents are the Biosafety in Microbiological and Biomedical Laboratories and NIH Guidelines for Research involving Recombinant or Synthetic Nucleic Acid Molecules. An effective OHSP generally requires the combined responsibility and cooperative interactions of several groups within an institution (Figure 1).9 A systematic and recurring review of the OHSP is essential to assessing its effectiveness and formulating action plans to correct shortcomings. OHSP evaluations by external experts (for example, those performed by AAALAC International during accreditation site visits) can also provide an institution with valuable feedback on whether its program meets or exceeds applicable standards. In fact, OHSP deficiencies have

A number of recent reports have documented likely swine-to-human virus transmission in swine facilities. During the month of January 2016, weekly bioaerosol and pig oral secretion samplings were performed in a pig handling facility to assess the possible occupational exposure to swine influenza A virus and adenovirus. During the 4 weeks, a total of 35 specimens were collected from multiple pig pens within the animal facility. One bioaerosol sample and five pig oral secretion samples were found positive for porcine adenovirus and further sequencing data revealed two different porcine adenoviruses. None of the samples showed evidence for influenza A virus by molecular assays. While swine adenoviruses are not thought to infect man, their detections suggests that bioaerosol sampling may be a non-invasive approach to detecting emergent zoonotic pathogens in agricultural industries.

China, Japan, and Korea have spent decades developing and amending laws, regulations, and guidelines to address the humane care and use of laboratory animals. This process began in 1983 in China, 1973 in Japan, and 1991 in Korea and has continued to the present. The governmental oversight of research varies between these countries, ranging from regulations by multiple levels of government in China to self-regulation under multiple government guidelines in Japan. Common to all is incorporation of the internationally recognized principles of the 3Rs: replacement, reduction and refinement. This paper reviews how the framework of laws, regulations, and guidelines evolved in each of these countries, their current status, and the expectation that they will continue to evolve.

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naivete mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 x 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.

Cynomolgus (or longtailed) macaques (Macaca fascicularis) are used extensively as laboratory animals in biomedical research. Their use in Singapore, an emerging biomedical hub in Southeast Asia, is now increasing widely, with research subjects currently originating from Singapore, Vietnam, and Pulau Bintan, Indonesia. Limited data exist on the genetic and phenotypic polymorphisms and phylogenetic relationships of these groups, and the animals are used as research subjects without regard to potential differences or homogeneity. Here we characterize their phenotypes by using established primatology tools to detail morphometrics and pelage erythrism and saturation. Pelage analyses supported the Gloger rule, in which heavily pigmented forms predominate near the equator, with Singaporean and Bintan macaques having darker pelage than Vietnamese macaques. Morphometric variation patterns suggest a tendency toward insular dwarfism and correlate generally with the Bergmann rule, in which body mass increases with latitude and colder climate. Although the 3 populations all belong to the nominotypical subspecies M.f.fascicularis, phenotypic differences are evident and are valuable tools to analyze their phylogeographic history and phylogenetic relationships.

Background A cynomolgus macaque (Macaca fascicularis) presented with decreased appetite, lethargy, ataxia, disorientation and visual impairment. It was used in a hepatitis B (HBV) study involving injections of HBV plasmid construct (450 mu g) and aflatoxin B1 (25 mu g/kg) in an effort to develop a model of liver cirrhosis and hepatocellular carcinoma.Methods The case work-up included physical examination, including assessment of hydration, thoracic and abdominal radiographs and abdominal ultrasound. Clinical pathology included complete blood counts and tests for levels of plasma ammonia (NH3) and serum electrolytes, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total bilirubin, and gamma-glutamyl transferase. Serum samples had also been serially tested for the presence of HBsAg, anti-HBc antibodies, HBV e-antigen, and HBV DNA. With a tentative diagnosis of hepatic encephalopathy, treatment with lactulose, antibiotics, and fluid therapy was initiated.Results Clinical pathology and diagnostic imaging performed on the animal revealed no abnormalities except for the hyperammoniemia. Absence of HBV markers in the serum indicated unsuccessful inoculation. Not fully responding to therapeutic intervention, the animal was euthanized. Necropsy revealed fibrous peritoneal adhesions and an abscess in the cerebellopontine angle. Exudate culture indicated the presence of alpha-hemolytic streptococcus, Eubacterium lentum, and Bacteroides stercoris.Conclusions Brain abscesses are uncommon in non-human primates. This case of cerebellar abscess is characterized by important features similar to that found in humans, including the presenting signs and the presence of the above-cultured bacteria.

Scientific Considerations and Choice of Species Regulatory Considerations in the Use of Animal Models Animal Handling and Surgical Procedures Basic Animal Investigative Methods Animal Welfare Considerations Safety Management of an Animal Facility Supporting Facilities Design The Development of Comprehensive Animal Facilities in Singapore.