Nonhuman Primate Model Research Articles

Adaptations to maternal WSD-feeding and metformin use on fetal islets from non-human primate offspring

Given the current increased prevalence of diabetes in pregnancy, the potential relative risks and benefits of oral glycemic agents on the developing offspring should be evaluated and considered. Metformin (Met) is a common oral glycemic agent prescribed in pregnancy that is not metabolized, but is renally excreted and actively transported across the placenta. Thus, the developing fetus is exposed to near equivalent maternal adult doses with subsequent risk of adverse consequences. Using a non-human primate model of maternal overnutrition, our group previously demonstrated that developmental exposure to a Western-style diet (WSD) results in an inappropriate increase in glucose-stimulated insulin secretion and changes in expression of several ion channels involved in insulin secretion in islets from juvenile offspring. Given that maternal overnutrition results in an excess energy supply to the developing fetus while Met inhibits mitochondrial respiration, we hypothesize that maternal WSD-feeding and Met will result in maladaptive beta-cell function in the fetus. Static insulin secretion assays on islets from chow (CD) and WSD +/- Met-exposed offspring show no differences in glucose-stimulated insulin secretion. Nevertheless, islets from a mWSD+Met-exposed fetus showed lower oxygen consumption and extracellular acidification rate relative to mWSD+placebo fetal islets, suggesting that maternal diet influences fetal programming and adaptation to Met in fetal islets. Therefore, we predict that the metabolic response to intrauterine Met exposure differs depending on the maternal diet. Elucidation of the mechanisms mediating adaptations to maternal diet and Met exposure during development may provide insight on interventions to avoid beta-cell failure in the offspring as they age. All nonhuman primate studies were supported in part by the Oregon National Primate Research Center grant P51 OD011092 from the National Institutes of Health/Offce of the Director. D.T.C. was supported by the Vanderbilt University Training Program in Molecular Endocrinology (5T32 DK563-30) and a predoctoral fellowship from the NIH/NIDDK (1F31 DK135164).T.A.D., C.E.M., K.M.A., J.E.F., P.K., and M.G. were supported by NIH/NIDDK Grants R24 DK090964 and R01 DK128187. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Does Rapamycin Treatment Alter Age-Associated Changes in Liver Proteome Profile of a Unique Non-Human Primate Model?

The common marmoset ( Callithrix jacchus) is a small, short-lived non-human primate, which has an analogous physiology to humans making it more suitable to elucidate mechanisms involved in the development of human diseases, particularly with aging. The objectives of this study were to identify for the first time whether 1) there are age-related changes in liver proteome profile of the marmoset, and 2) these changes can be reversed by rapamycin, a drug known to slow aging in rodents. We based these objectives on our own previously observed significant age-associated changes in specific lipid classes in serum and liver tissues of marmosets. We hypothesized that the liver proteome of marmosets will be altered with age, and treatment with rapamycin will prevent many of these age-associated changes. To test our hypothesis, we performed liver tissue proteomics to identify proteins that change with age. Our data reveal that the liver proteome of the old untreated marmosets is distinct compared to that of the young untreated or the old animals treated with rapamycin. Interestingly, we find a significant increase in the enzyme ceramidase (ASAH1) in old marmosets treated with rapamycin compared to the old untreated animals. Rapamycin treatment increased ceramidase levels in old marmosets to that observed in young untreated animals. Ceramidase mediates the hydrolysis of ceramides. Our lipidomic results previously revealed that there is an age-associated increase in hepatic ceramides which is significantly reduced upon treatment of marmosets with rapamycin. Accumulation of ceramides has been linked to insulin resistance, mitochondrial dysfunction, inflammation, and activation of triglyceride storage which results in the development and progression of diseases like nonalcoholic fatty liver disease (NAFLD). Taken together, our results suggest that treatment with rapamycin increases hepatic ceramidase levels which may reduce age-associated increases in ceramide levels and consequently lead to improved hepatic function and reduced lipid accumulation. Further analysis of the hepatic proteome profile revealed a decrease in choline phosphotransferase (CHPT1) and fetuin A in young untreated animals or old animals treated with rapamycin compared to old untreated animals. Choline phosphotransferase (CHPT1) catalyzes the synthesis of phosphatidylcholine which is a regulator of cell membrane integrity while fetuin A is a proinflammatory protein produced by hepatocytes. Both these proteins also play important roles in the development of NAFLD. We are now performing pathway analysis to narrow down the pathways that may play an important role in age-associated hepatic fat accumulation, a critical step in the development of NAFLD. This work was supported by NIA/NIH 1R21 AG067164-01 (AK, AS) and a NIA/NIH P30AG044271 San Antonio Claude D. Pepper Older Americans Independence Center, PESC Pilot Grant Award (AK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Alterations in adolescent brain serotonin (5HT)1A, 5HT2A, and dopamine (D)2 receptor systems in a nonhuman primate model of early life adversity.

Stress affects brain serotonin (5HT) and dopamine (DA) function, and the effectiveness of 5HT and DA to regulate stress and emotional responses. However, our understanding of the long-term impact of early life adversity (ELA) on primate brain monoaminergic systems during adolescence is scarce and inconsistent. Filling this gap in the literature is critical, given that the emergence of psychopathology during adolescence has been related to deficits in these systems. Here, we use a translational nonhuman primate (NHP) model of ELA (infant maltreatment by the mother) to examine the long-term impact of ELA on adolescent 5HT1A, 5HT2A and D2 receptor systems. These receptor systems were chosen based on their involvement in stress/emotional control, as well as reward and reinforcement. Rates of maternal abuse, rejection, and infant's vocalizations were obtained during the first three postnatal months, and hair cortisol concentrations obtained at 6 months postnatal were examined as early predictors of binding potential (BP) values obtained during adolescence using positron emission tomography (PET) imaging. Maltreated animals demonstrated significantly lower 5HT1A receptor BP in prefrontal cortical areas as well as the amygdala and hippocampus, and lower 5HT2A receptor BP in striatal and prefrontal cortical areas. Maltreated animals also demonstrated significantly lower D2 BP in the amygdala. None of the behavioral and neuroendocrine measurements obtained early in life predicted any changes in BP data. Our findings suggest that early caregiving experiences regulate the development of brain 5HT and DA systems in primates, resulting in long-term effects evident during adolescence.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = −0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Genetic characterization of a captive marmoset (Callithrix jacchus) colony using genotype-by-sequencing.

The marmoset is a fundamental nonhuman primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Complete Genomic Assembly of Mauritian Cynomolgus Macaque Killer Ig-like Receptor and Natural Killer Group 2 Haplotypes.

Mauritian-origin cynomolgus macaques (MCMs) serve as a powerful nonhuman primate model in biomedical research due to their unique genetic homogeneity, which simplifies experimental designs. Despite their extensive use, a comprehensive understanding of crucial immune-regulating gene families, particularly killer Ig-like receptors (KIR) and NK group 2 (NKG2), has been hindered by the lack of detailed genomic reference assemblies. In this study, we employ advanced long-read sequencing techniques to completely assemble eight KIR and seven NKG2 genomic haplotypes, providing an extensive insight into the structural and allelic diversity of these immunoregulatory gene clusters. Leveraging these genomic resources, we prototype a strategy for genotyping KIR and NKG2 using short-read, whole-exome capture data, illustrating the potential for cost-effective multilocus genotyping at colony scale. These results mark a significant enhancement for biomedical research in MCMs and underscore the feasibility of broad-scale genetic investigations.

Sex-Dependent Effects on Influenza-Specific Antibody Quantity and Neutralizing Activity following Vaccination of Newborn Non-Human Primates Is Determined by Adjuvants.

A number of studies have demonstrated the role of sex in regulating immune responses to vaccination. However, these findings have been limited to adults for both human and animal models. As a result, our understanding of the impact of sex on vaccine responses in the newborn is highly limited. Here, we probe this important question using a newborn non-human primate model. We leveraged our prior analysis of two cohorts of newborns, with one being mother-reared and one nursery-reared. This provided adequate numbers of males and females to interrogate the impact of sex on the response to inactivated influenza vaccines alone or adjuvanted with R848, flagellin, or both. We found that, in contrast to what has been reported in adults, the non-adjuvanted inactivated influenza virus vaccine induced similar levels of virus-specific IgG in male and female newborns. However, the inclusion of R848, either alone or in combination with flagellin, resulted in higher antibody titers in females compared to males. Sex-specific increases in the neutralizing antibody were only observed when both R848 and flagellin were present. These data, generated in the highly translational NHP newborn model, provide novel insights into the role of sex in the immune response of newborns.

Infection with SARS-CoV-2 can cause pancreatic impairment

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (β) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor β. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

B-cell–directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates

AbstractChimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell–derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell–targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor–independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell–targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: candidate selection in a preclinical murine model.

In the early COVID-19 pandemic with urgent need for countermeasures, we aimed at developing a replicating viral vaccine using the highly efficacious measles vaccine as vector, a promising technology with prior clinical proof of concept. Building on our successful pre-clinical development of a measles virus (MV)-based vaccine candidate against the related SARS-CoV, we evaluated several recombinant MV expressing codon-optimized SARS-CoV-2 spike glycoprotein. Candidate V591 expressing a prefusion-stabilized spike through introduction of two proline residues in HR1 hinge loop, together with deleted S1/S2 furin cleavage site and additional inactivation of the endoplasmic reticulum retrieval signal, was the most potent in eliciting neutralizing antibodies in mice. After single immunization, V591 induced similar neutralization titers as observed in sera of convalescent patients. The cellular immune response was confirmed to be Th1 skewed. V591 conferred long-lasting protection against SARS-CoV-2 challenge in a murine model with marked decrease in viral RNA load, absence of detectable infectious virus loads, and reduced lesions in the lungs. V591 was furthermore efficacious in an established non-human primate model of disease (see companion article [S. Nambulli, N. Escriou, L. J. Rennick, M. J. Demers, N. L. Tilston-Lunel et al., J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23]). Thus, V591 was taken forward into phase I/II clinical trials in August 2020. Unexpected low immunogenicity in humans (O. Launay, C. Artaud, M. Lachâtre, M. Ait-Ahmed, J. Klein et al., eBioMedicine 75:103810, 2022, https://doi.org/10.1016/j.ebiom.2021.103810) revealed that the underlying mechanisms for resistance or sensitivity to pre-existing anti-measles immunity are not yet understood. Different hypotheses are discussed here, which will be important to investigate for further development of the measles-vectored vaccine platform.IMPORTANCESARS-CoV-2 emerged at the end of 2019 and rapidly spread worldwide causing the COVID-19 pandemic that urgently called for vaccines. We developed a vaccine candidate using the highly efficacious measles vaccine as vector, a technology which has proved highly promising in clinical trials for other pathogens. We report here and in the companion article by Nambulli et al. (J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23) the design, selection, and preclinical efficacy of the V591 vaccine candidate that was moved into clinical development in August 2020, 7 months after the identification of SARS-CoV-2 in Wuhan. These unique in-human trials of a measles vector-based COVID-19 vaccine revealed insufficient immunogenicity, which may be the consequence of previous exposure to the pediatric measles vaccine. The three studies together in mice, primates, and humans provide a unique insight into the measles-vectored vaccine platform, raising potential limitations of surrogate preclinical models and calling for further refinement of the platform.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

A wireless, implantable bioelectronic system for monitoring urinary bladder function following surgical recovery

Partial cystectomy procedures for urinary bladder-related dysfunction involve long recovery periods, during which urodynamic studies (UDS) intermittently assess lower urinary tract function. However, UDS are not patient-friendly, they exhibit user-to-user variability, and they amount to snapshots in time, limiting the ability to collect continuous, longitudinal data. These procedures also pose the risk of catheter-associated urinary tract infections, which can progress to ascending pyelonephritis due to prolonged lower tract manipulation in high-risk patients. Here, we introduce a fully bladder-implantable platform that allows for continuous, real-time measurements of changes in mechanical strain associated with bladder filling and emptying via wireless telemetry, including a wireless bioresorbable strain gauge validated in a benchtop partial cystectomy model. We demonstrate that this system can reproducibly measure real-time changes in a rodent model up to 30 d postimplantation with minimal foreign body response. Studies in a nonhuman primate partial cystectomy model demonstrate concordance of pressure measurements up to 8 wk compared with traditional UDS. These results suggest that our system can be used as a suitable alternative to UDS for long-term postoperative bladder recovery monitoring.

Development and characterization of a non-human primate model of disseminated synucleinopathy.

The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

Quantitative and Qualitative Distinctions between HIV-1 and SIV Reservoirs: Implications for HIV-1 Cure-Related Studies.

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Abstract 6312: Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against blood malignancies. However, prominent barriers, including limited proliferation, CAR T cell persistence, and poor T cell effector function, hinder its potential, especially in solid tumors. Various engineering approaches attempt to address these issues by armoring CAR T cells with factors that enhance the function and maintenance of T cells in the anti-tumor response. We previously described a human orthogonal IL-2/IL-2R system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (oRβ) that does not significantly respond to its native ligand, wild type IL-2. This system provides a “private IL-2 signal” in engineered oRβ-expressing cells while avoiding signaling in bystander immune cells, reducing toxicities. We have demonstrated STK-009/oRβ’s ability to selectively expand oRβ-expressing CAR T cells (SYNCAR T cells) targeting heme (SYNCAR-001, CD19) and solid tumor (SYNCAR-002, GPC3) targets at will. Importantly, the system enhanced anti-tumor control driving deep and durable responses in both bulky lymphoma and hepatocellular carcinoma (HCC) tumor models. Additionally, in a non-human primate model, STK-009 did not potentiate wild type T or NK cells and exhibited no evidence of toxicity. Currently, the combination of STK-009 + SYNCAR-001 is undergoing Phase I clinical trials (NCT05665062). Here, we conducted a direct comparison of several armoring approaches including the SYNCAR platform, the overexpression of c-Jun, an IL-15/IL-15Rα fusion, and IL-18 in human CAR T cells targeting GPC3. To assess their effects in overcoming CAR T cell dysfunction, cells were subjected to continuous antigen exposure in vitro. STK-009 treated SYNCAR-002 T cells displayed an enhanced proliferative capacity, sustained effector cytokine secretion, and an increased activation state when compared to other armoring technologies and PBS treatment. In a HCC tumor model, treatment with STK-009/SYNCAR-002 demonstrated superior tumor control with minimal toxicities. Interestingly, IL-18 overexpression also controlled tumors, but was accompanied by uncontrolled CAR T cell expansion and toxicity resembling severe GVHD. scRNAseq analyses revealed STK-009-treated SYNCAR-002 T cells simultaneously activated effector genes and repressed an exhaustion gene signature. This analysis also identified a novel mechanism by which STK-009/oRβ signaling induces a superior synthetic effector state via transcriptional repression of the stress response/proteosome. These findings support the advantages of an orthogonal platform that selectively drives and sustains potent T cell effector function without safety concerns associated with constitutively expressed armored approaches. Citation Format: Ethan Jung, Helena Silva, Marie Semana, Ivan Cheng, Somya Singh, Michele Bauer, Mohammed Ali, Henry Rosas, Ryan Burgess, George Zeng, Woei Chang, Navneet Ratti, Deepti Rokkam, Patrick J. Lupardus, Martin Oft, Paul-Joseph Aspuria. Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6312.