Large animal model species in pluripotent stem cell therapy research and development for retinal diseases: a systematic review.

Aim: Retinal cell therapy modalities, in the category of advanced therapy medicinal products (ATMPs), are being developed to target several retinal diseases. Testing in large animal models (LAMs) is a crucial step in translating retinal ATMPs into clinical practice. However, challenges including budgetary and infrastructure constraints can hinder LAM research design and execution. Here, to facilitate the comparison of the various LAMs in pluripotent retinal cell therapy research, we aimed to systematically evaluate the species distribution, reported scientific utility, and methodology of a range of LAMs. Methods: A systematic search using the words retina, stem cell, transplantation, large animal, pig, rabbit, dog, and nonhuman primate was conducted in the PubMed, Embase, Science Direct and GoogleScholar databases in February 2023. Results: We included 22 studies involving pluripotent stem cells (induced pluripotent stem cells or human embryonic stem cells) in LAMs, including non-human primates (NHP), pigs, dogs, and rabbits. Nearly half of the studies utilized wild-type animal models. In other studies, retinal degeneration features were simulated via laser, chemical, or genetic insult. Transplants were delivered subretinally, either as cell suspensions or pre-formed monolayers (with or without biodegradable scaffolding). The transplanted cells dose per eye varied widely (40,000 - 4,000,000 per dose). Cells were delivered via vitrectomy surgery in 15 studies and by an "ab externo" approach in one study. Structural outcomes were assessed using confocal scanning laser ophthalmoscopy imaging. Functional outcomes included multifocal electroretinogram and, in one case, a measure of visual acuity. Generally, cell suspension transplants exhibited low intraretinal incorporation, while monolayer transplants incorporated more efficiently. Immune responses posed challenges for allogeneic transplants, suggesting that autologous iPSC-derived transplants may be required to decrease the likelihood of rejection. Conclusion: The use of appropriate LAMs helps to advance the development of retinal ATMPs. The anatomical similarity of LAM and human eyes allows the implementation of clinically-relevant surgical techniques. While the FDA Modernization Act 2.0 has provided a framework to consider alternative methods including tissue-on-a-chip and human cell culture models for pharmacologic studies, LAM testing remains useful for cell and tissue replacement studies to inform the development of clinical trial protocols.

Determination of an Acclimation Period for Swine in Biomedical Research.

Swine are widely used models in biomedical research due to their physiologic and anatomic similarities to humans. During transport from vendors to research facilities, pigs are subject to a number of stressors, including environmental, social, and stress as a result of deprivation from food and water. As stress can have a number of adverse psychologic and physiologic effects, an acclimation period, defined as the period of time that an animal has to adjust and stabilize in a new environment, is recommended. The literature indicates that swine should be conditioned to their new facility for 5 to 7 d prior to undergoing survival surgery; however, to date, there is no published scientific evidence to support this or any specific acclimation period for swine. To investigate whether a certain length acclimation period leads to decreased stress in swine, we measured 2 stress biomarkers, cortisol and chromogranin A (CgA), from the saliva of 12 naive Yorkshire swine (n = 6 males and 6 females) arriving at our facility for use in research protocols. Noninvasive saliva collection was performed on days 1, 3, 5, 7, 10, and 14 after arrival from the vendor (representing different acclimation periods). We hypothesized that longer acclimation periods would result in reduced levels of both cortisol and CgA, indicating reduced stress. Our data revealed that there was no statistical difference in cortisol levels over time (P = 0.8200), nor between the sexes (P = 0.9886) or individual animals (P = 0.6280). CgA, similarly to cortisol, showed no overall effect of time (P = 0.2017) or sex (P = 0.6598). For this analyte, individual animal was significant (P < 0.0001), which suggests high interanimal variation. Furthermore, there was a significant decrease (P = 0.0077) in salivary CgA from day 1 compared with day 14, suggesting that swine may benefit from an acclimation period of at least 14 d.

Amelioration of airway and GI disease in G551D-CF ferrets by AAV1 and AAV6.

Gene therapy for CF has concentrated on targeting the lung. Here we took a different approach by injecting into the cephalic vein and spraying into the trachea of G551D, CF ferrets either AAV1 or 6 containing Δ27-264-CFTR, a truncated version of CFTR. Treatment with the potentiator VX-770 was halted for 7 days before instillation to induce a disease phenotype. Indeed, all ferrets were pancreas-insufficient when they entered the study. Four ferrets (three receiving AAV1 and one AAV6) were necropsied 48 days after vector delivery, and four (three receiving AAV6, one AAV1) were euthanized or died prior to the planned necropsy. AAV1 or AAV6 vector genomes, mRNA expression, and CFTR protein were detected in all tracheal and lung samples and in the liver, pancreas, and ileum of the treated ferrets. Surface and basal airway cells, pancreatic and bile ducts, and ileal crypts and villi were successfully transduced. Obstruction of the airways accompanied by pulmonary hemorrhaging, plugged pancreatic and bile ducts as well as mucous plugs in the ileum were noticed in untreated but absent from transduced ferrets necropsied at 48 days. Transduction of G551D ferrets suggests that a combination of systemic and airway application may be the preferred route of delivery for CF.

The Use of Guanfacine to Mediate Anxiety-related Reactivity and Reduce Associated Agonistic Behavior in Two Pigtail Macaques (Macaca nemestrina).

Guanfacine, an α 2adrenoceptor agonist, has been used to successfully treat self-injurious behavior in nonhuman primates, including macaques (Macaca mulatta) and baboons (Papio anubis). It does so by facilitating a correction to the dopaminergic system that mediates a reduction in impulsivity and reactivity. Given this, we assessed the potential efficacy of guanfacine to treat socially directed agonistic behavior in primates with an apparent reactive behavioral phenotype. We present data from 2 pigtail macaques (Macaca nemestrina): an intact adult male housed in a breeding group, and an experimentally naive adult female living in a research setting with her social partner. Baseline behavioral assessments suggested that both macaques showed extreme responses to external stressors that triggered them to aggress social partners often leading to wounding that required veterinary intervention. Both animals were tracked during the course of 1 y. Once treated regularly with guanfacine, both animals showed significant reduction in their agonistic behavior and the rate at which they wounded other animals. Indeed, in the year since the female has been treated with guanfacine she has never wounded her cagemate. By collecting regular and detailed behavioral observations on the male in the breeding colony, we were able to identify triggers for his aggression and to track the behavioral changes evidenced after guanfacine treatment. These data supported our hypothesis that his aggression reflected extreme reactivity to external stressors, rather than general anxiety. Importantly, we saw only a limited and short-lived reduction in the male's affiliative behavioral rates, and thus guanfacine had no sedative effect, but did successfully reduce his reactivity and resultant agonism and wounding.

Comparing different strategies to reduce hepatocellular damage in obese common marmosets (Callithrix jacchus).

BACKGROUND: Obesity in common marmosets (Callithrix jacchus) can lead to various liver pathologies. In other species, reduced caloric intake and weight loss improve prognosis, and, often, hepatoprotectants are used to halt or reverse hepatocellular damage from fat deposition in the liver. There are no published therapies for reducing hepatocellular damage in obese marmosets. METHODS: Fifteen obese marmosets were used to evaluate the ability of caloric restriction and pharmacologic therapy (S-adenosylmethionine + milk thistle extract, or SMT), alone and combined, to reduce elevated liver enzymes. Body weight and serum chemistries were measured every 4 weeks for 6 months. RESULTS: Across treatment groups, there was a significant reduction in liver enzymes ALT and AST over time. SMT alone significantly reduced liver enzymes ALT and AST at 6 months from baseline. CONCLUSIONS: Caloric restriction and SMT, alone and combined, are effective at reducing liver enzyme levels in obese marmosets.

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood.

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 h post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labelled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

Transduction of Ferret Surface and Basal Cells of Airways, Lung, Liver, and Pancreas via Intratracheal or Intravenous Delivery of Adeno-Associated Virus 1 or 6.

Cystic fibrosis (CF) is potentially treatable by gene therapy. Since the identification of the CF gene, preclinical and clinical trials have concentrated on achieving effective gene therapy targeting the lung. However, the lung has proven to be a formidable barrier to successful gene therapy especially for CF, and many clinical trials failed to achieve efficacy. Recent advances in vector design and adeno-associated virus (AAV) serotypes have increased the chances of success. Given that CF is a multi-organ disease, the goal of this study was to test whether a gene therapy approach involving AAV1 or AAV6 vector delivery via the systemic circulation would at the same time overcome the barrier of lung delivery and transduce organs commonly affected by CF. To accomplish this, we sprayed AAV1 containing green fluorescent protein (GFP) into the trachea or injected it intravenously (IV). We also tested AAV6 injected IV. No adverse events were noted. Ferrets were necropsied 30 days after vector delivery. AAV1 or AAV6 vector genomes, messenger RNA (mRNA) expression, and GFP were detected in all the tracheal and lung samples from the treated animals, whether AAV1 was sprayed into the trachea or injected IV or AAV6 was injected IV. Importantly, both surface epithelial and basal cells of the trachea and lung airways were successfully transduced, regardless of which route of delivery or vector serotype used for transduction. We detected also AAV1 and AAV6 vector genomes, mRNA expression, and GFP in the livers and pancreases, particularly in the acinar cells of the pancreatic duct. These data suggest that gene transfer is attainable in the airways, liver, and pancreas using either serotype, AAV1 or AAV6. Given that these same organs are affected in CF, systemic delivery of AAV may be the preferred route of delivery for a gene therapy for CF.

Geometric Mismatch Promotes Anatomic Repair in Periorbital Bony Defects in Skeletally Mature Yucatan Minipigs.

Porous tissue-engineered 3D-printed scaffolds are a compelling alternative to autografts for the treatment of large periorbital bone defects. Matching the defect-specific geometry has long been considered an optimal strategy to restore pre-injury anatomy. However, studies in large animal models have revealed that biomaterial-induced bone formation largely occurs around the scaffold periphery. Such ectopic bone formation in the periorbital region can affect vision and cause disfigurement. To enhance anatomic reconstruction, geometric mismatches are introduced in the scaffolds used to treat full thickness zygomatic defects created bilaterally in adult Yucatan minipigs. 3D-printed, anatomically-mirrored scaffolds are used in combination with autologous stromal vascular fraction of cells (SVF) for treatment. An advanced image-registration workflow is developed to quantify the post-surgical geometric mismatch and correlate it with the spatial pattern of the regenerating bone. Osteoconductive bone growth on the dorsal and ventral aspect of the defect enhances scaffold integration with the native bone while medio-lateral bone growth leads to failure of the scaffolds to integrate. A strong positive correlation is found between geometric mismatch and orthotopic bone deposition at the defect site. The data suggest that strategic mismatch >20% could improve bone scaffold design to promote enhanced regeneration, osseointegration, and long-term scaffold survivability.

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model.

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in >100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

Bacterial Cholecystitis and Cholangiohepatitis in Common Marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus), a New World NHP, has emerged as important animal model in multiple areas of translational biomedical research. The quality of translational research in marmosets depends on early diagnosis, treatment, and prevention of their spontaneous diseases. Here, we characterize an outbreak of infectious cholangiohepatitis that affected 7 adult common marmosets in a single building over a 10-mo period. Marmosets presented for acute onset of lethargy, dull mentation, weight loss, dehydration, hyporexia, and hypothermia. Blood chemistries at presentation revealed markedly elevated hepatic and biliary enzymes, but mild neutrophilia was detected in only 1 of the 7. Affected marmosets were unresponsive to rigorous treatment and died or were euthanized within 48 h of presentation. Gross and histopathologic examinations revealed severe, necrosuppurative cholangiohepatitis and proliferative cholecystitis with bacterial colonies and an absence of gallstones. Perimortem and postmortem cultures revealed single or dual isolates of Escherichia coli and Pseudomonas aeruginosa. Other postmortem findings included bile duct hyperplasia, periportal hepatitis, bile peritonitis, ulcerative gastroenteritis, and typhlitis. Environmental contamination of water supply equipment with Pseudomonas spp. was identified as the source of infection, but pathogenesis remains unclear. This type of severe, infectious cholangiohepatitis with proliferative cholecystitis with Pseudomonas spp. had not been reported previously in marmosets, and we identified and here describe several contributing factors in addition to contaminated drinking water.