Genetic barrier to resistance: a critical parameter for efficacy of neutralizing monoclonal antibodies against SARS-CoV-2 in a nonhuman primate model.

The potency of antibody neutralization in cell culture has been used as the key criterion for selection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for clinical development. As other aspects may also influence the degree of protection in vivo, we compared the efficacy of two neutralizing monoclonal antibodies (TRES6 and 4C12) targeting different epitopes of the receptor binding domain (RBD) of SARS-CoV-2 in a prophylactic setting in rhesus monkeys. All four animals treated with TRES6 had reduced viral loads in the upper respiratory tract 2 days after naso-oropharyngeal challenge with the Alpha SARS-CoV-2 variant. Starting 2 days after challenge, mutations conferring resistance to TRES6 were dominant in two of the rhesus monkeys, with both animals failing to maintain reduced viral loads. Consistent with its lower serum neutralization titer at the day of challenge, prophylaxis with 4C12 tended to suppress viral load at day 2 less efficiently than TRES6. However, a week after challenge, mean viral loads in the lower respiratory tract in 4C12-treated animals were lower than in the TRES6 group and no mutations conferring resistance to 4C12 could be detected in viral isolates from nasal or throat swabs. Thus, genetic barrier to resistance seems to be a critical parameter for the efficacy of prophylaxis with monoclonal antibodies against SARS-CoV-2. Furthermore, comparison of antibody concentrations in respiratory secretions to those in serum shows reduced distribution of the 4C12 antibody into respiratory secretions and a delay in the appearance of antibodies in bronchoalveolar lavage fluid compared to their appearance in secretions of the upper respiratory tract.IMPORTANCEMonoclonal antibodies are a powerful tool for the prophylaxis and treatment of acute viral infections. Hence, they were one of the first therapeutic agents licensed for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oftentimes, the main criterion for the selection of antibodies for clinical development is their potency of neutralization in cell culture. By comparing two antibodies targeting the Spike protein of SARS-CoV-2, we now observed that the antibody that neutralized SARS-CoV-2 more efficiently in cell culture suppressed viral load in challenged rhesus monkeys to a lesser extent. Extraordinary rapid emergence of mutants of the challenge virus, which had lost their sensitivity to the antibody, was identified as the major reason for the reduced efficacy of the antibody in rhesus monkeys. Therefore, the viral genetic barrier to resistance to antibodies also affects their efficacy.

Stem cell-derived CAR T cells show greater persistence, trafficking, and viral control compared to ex vivo transduced CAR T cells.

Adoptive cell therapy (ACT) using T cells expressing chimeric antigen receptors (CARs) is an area of intense investigation in the treatment of malignancies and chronic viral infections. One of the limitations of ACT-based CAR therapy is the lack of in vivo persistence and maintenance of optimal cell function. Therefore, alternative strategies that increase the function and maintenance of CAR-expressing T cells are needed. In our studies using the humanized bone marrow/liver/thymus (BLT) mouse model and nonhuman primate (NHP) model of HIV infection, we evaluated two CAR-based gene therapy approaches. In the ACT approach, we used cytokine enhancement and preconditioning to generate greater persistence of anti-HIV CAR+ T cells. We observed limited persistence and expansion of anti-HIV CAR T cells, which led to minimal control of the virus. In our stem cell-based approach, we modified hematopoietic stem/progenitor cells (HSPCs) with anti-HIV CAR to generate anti-HIV CAR T cells in vivo. We observed CAR-expressing T cell expansion, which led to better plasma viral load suppression. HSPC-derived CAR cells in infected NHPs showed superior trafficking and persistence in multiple tissues. Our results suggest that a stem cell-based CAR T cell approach may be superior in generating long-term persistence and functional antiviral responses against HIV infection.

A nonhuman primate model of blue light-induced progressive outer retina degeneration showing brimonidine drug delivery system-mediated cyto- and neuroprotection.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by atrophy of the retinal pigment epithelium (RPE), loss of photoreceptors, and disruption of choriocapillaris. Excessive light exposure is toxic to the retina and is a known risk factor for AMD. We first investigated the effects of blue light-induced phototoxicity on RPE and photoreceptors in nonhuman primates (NHPs, a model of progressive retinal degeneration) and then evaluated the potential cyto- and neuroprotective effects of the brimonidine drug delivery system (Brimo DDS). In the first set of experiments related to model development, parafoveal lesions of varying severity were induced using blue light irradiation of the retina of cynomolgus monkeys to evaluate the level of phototoxicity in the RPE and photoreceptors. RPE damage was assessed using fundus autofluorescence imaging to quantify areas of hypofluorescence, while thinning of the outer nuclear layer (ONL, photoreceptor nuclei) was quantified using optical coherence tomography (OCT). Photoreceptor function was assessed using multifocal electroretinography (mfERG). RPE damage progressively increased across all lesion severities from 2 to 12 weeks, as did the extent of ONL thinning. Lesions of high severity continued to show reduction in mfERG amplitude, reaching a statistically significant maximum reduction at 12 weeks. Collectively, the first set of experiments showed that blue light irradiation of the NHP eye resulted in progressive retinal degeneration identified by damage to RPE, ONL thinning, and disrupted photoreceptor function - hallmarks of GA in humans. We then used the model to evaluate the cyto- and neuroprotective effects of Brimo DDS, administered as a therapeutic after allowing the lesions to develop for 5 weeks. Placebo DDS or Brimo DDS were administered intravitreally and a set of untreated animals were used as an additional control. In the placebo DDS group, hypofluorescence area continued to increase from baseline, indicating progressive RPE damage, while progression was significantly slowed in eyes receiving Brimo DDS. Likewise, ONL thinning continued to progress over time in eyes that received the placebo DDS, but was reduced in Brimo DDS-treated eyes. Pharmacologically relevant brimonidine concentrations were sustained in the retina for up to 26 weeks following Brimo DDS administration. In summary, Brimo DDS demonstrated cyto- and neuroprotective effects in a novel NHP GA model of progressive retinal degeneration.

Biomimetic surgical mesh to replace fascia with tunable force-displacement.

Here we mimic the mechanical properties of native fascia to design surgical mesh for fascia replacement. Despite the widespread acceptance of synthetic materials as tissue scaffolds for pelvic floor disorders, mechanical property mismatch between mesh and adjacent native tissue drives fibrosis and erosion, leading the FDA to remove several surgical meshes from the market. However, autologous tissue does not induce either fibrosis or adjacent tissue erosion, suggesting the potential for biomimetic surgical mesh. In this study, we determined the design rules for mesh that mimics native fascia by mathematically modeling multi-component polymer networks, composed of elastin-like and collagen-like fibers, using a spring-network model. To validate the model, we measured the stress-strain curves of native bovine and nonhuman primate (Macaca mulatta) abdominal fascia in both toe and linear regions. We find that the stiffer collagen-like fibers must remain limp until the elastin-like fibers extend to the initial length of spanning collagen-like fibers under uniaxial tension. Comparing model results to experiment determines the product of fiber volume fraction and elastic modulus, a critical design parameter. Dual fiber mesh with mechanical properties that mimic fascia are feasible. These results have broad application to a wide range of soft tissue replacements including ~200,000 surgeries/year for pelvic floor disorders, because standard-of-care mesh contain only stiffer polymers that behave more like collagen than native tissue.

Bardet-Biedl Syndrome in rhesus macaques: A nonhuman primate model of retinitis pigmentosa.

The development of therapies for retinal disorders is hampered by a lack of appropriate animal models. Higher nonhuman primates are the only animals with retinal structure similar to humans, including the presence of a macula and fovea. However, few nonhuman primate models of genetic retinal disease are known. We identified a lineage of rhesus macaques with a frameshift mutation in exon 3 of the BBS7 gene c.160delG (p.Ala54fs) that is predicted to produce a non-functional protein. In humans, mutations in this and other BBS genes cause Bardet-Biedl syndrome, a ciliopathy and a syndromic form of retinitis pigmentosa generally occurring in conjunction with kidney dysfunction, polydactyly, obesity, and/or hypogonadism. Three full- or half-sibling monkeys homozygous for the BBS7 c.160delG variant, at ages 3.5, 4 and 6 years old, displayed a combination of severe photoreceptor degeneration and progressive kidney disease. In vivo retinal imaging revealed features of severe macular degeneration, including absence of photoreceptor layers, degeneration of the retinal pigment epithelium, and retinal vasculature atrophy. Electroretinography in the 3.5-year-old case demonstrated loss of scotopic and photopic a-waves and markedly reduced and delayed b-waves. Histological assessments in the 4- and 6-year-old cases confirmed profound loss of photoreceptors and inner retinal neurons across the posterior retina, with dramatic thinning and disorganization of all cell layers, abundant microglia, absent or displaced RPE cells, and significant gliosis in the subretinal space. Retinal structure, including presence of photoreceptors, was preserved only in the far periphery. Ultrasound imaging of the kidneys revealed deranged architecture, and renal histopathology identified distorted contours with depressed, fibrotic foci and firmly adhered renal capsules; renal failure occurred in the 6-year-old case. Magnetic resonance imaging obtained in one case revealed abnormally low total brain volume and unilateral ventricular enlargement. The one male had abnormally small testes at 4 years of age, but polydactyly and obesity were not observed. Thus, monkeys homozygous for the BBS7 c.160delG variant closely mirrored several key features of the human BBS syndrome. This finding represents the first identification of a naturally-occurring nonhuman primate model of BBS, and more broadly the first such model of retinitis pigmentosa and a ciliopathy with an associated genetic mutation. This important new preclinical model will provide the basis for better understanding of disease progression and for the testing of new therapeutic options, including gene and cell-based therapies, not only for BBS but also for multiple forms of photoreceptor degeneration.

Establishment of cell lines derived from the genus Macaca through controlled expression of cell cycle regulators.

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.

Comprehensive Assessment of Ischemic Stroke in Nonhuman Primates: Neuroimaging, Behavioral, and Serum Proteomic Analysis.

Ischemic strokes, prevalence and impactful, underscore the necessity of advanced research models closely resembling human physiology. Our study utilizes nonhuman primates (NHPs) to provide a detailed exploration of ischemic stroke, integrating neuroimaging data, behavioral outcomes, and serum proteomics to elucidate the complex interplay of factors involved in stroke pathophysiology. We observed a consistent pattern in infarct volume, peaking at 1-month postmiddle cerebral artery occlusion (MCAO) and then stabilized. This pattern was strongly correlated to notable changes in motor function and working memory performance. Using diffusion tensor imaging (DTI), we detected significant alterations in fractional anisotropy (FA) and mean diffusivity (MD) values, signaling microstructural changes in the brain. These alterations closely correlated with the neurological and cognitive deficits that we observed, highlighting the sensitivity of DTI metrics in stroke assessment. Behaviorally, the monkeys exhibited a reliance on their unaffected limb for compensatory movements, a common response to stroke impairment. This adaptation, along with consistent DTI findings, suggests a significant impact of stroke on motor function and spatial perception. Proteomic analysis through MS/MS functional enrichment identified two distinct groups of proteins with significant changes post-MCAO. Notably, MMP9, THBS1, MB, PFN1, and YWHAZ were identified as potential biomarkers and therapeutic targets for ischemic stroke. Our results underscore the complex nature of stroke and advocate for an integrated approach, combining neuroimaging, behavioral studies, and proteomics, for advancing our understanding and treatment of this condition.

Nonnegligible Contribution of Nonlymphoid Tissue to Viral Reservoir During the Short-Term Early cART in SIVmac239-Infected Chinese Rhesus Macaques.

HIV/AIDS cannot be cured because of the persistence of the viral reservoir. Because of the complexity of the cellular composition and structure of the human organs, HIV reservoirs of anatomical site are also complex. Recently, although a variety of molecules have been reported to be involved in the establishment and maintenance of the viral reservoirs, or as marker of latent cells, the research mainly focuses on blood and lymph nodes. Now, the characteristics of the viral reservoir in tissue are not yet fully understood. In this study, various tissues were collected from SIVmac239-infected monkeys, and the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in them were compared with character of the anatomical viral reservoir under early treatment. The results showed that short-term combination antiretroviral therapy (cART) starting from 3 days after infection could significantly inhibit viremia and reduce the size of the anatomical viral reservoir, but it could not eradicate de novo infections and ongoing replication of virus. Moreover, the effects of early cART on the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in different tissues were different, which changed the size distribution of viral reservoir in anatomical site. Finally, the contribution of nonlymphoid tissues, especially liver and lung, to the viral reservoir increased after treatment, while the contribution of intestinal lymphoid to the viral reservoir significantly reduced. These results suggested that early treatment effectively decreased the size of viral reservoir, and that the effects of cART on the tissue viral reservoir varied greatly by tissue type. The results implied that persistent existence of virus in nonlymphoid tissues after short-term treatment suggested that the role of nonlymphoid tissues cannot be ignored in development strategies for AIDS therapy.

Genetic regulatory effects in response to a high-cholesterol, high-fat diet in baboons.

Steady-state expression quantitative trait loci (eQTLs) explain only a fraction of disease-associated loci identified through genome-wide association studies (GWASs), while eQTLs involved in gene-by-environment (GxE) interactions have rarely been characterized in humans due to experimental challenges. Using a baboon model, we found hundreds of eQTLs that emerge in adipose, liver, and muscle after prolonged exposure to high dietary fat and cholesterol. Diet-responsive eQTLs exhibit genomic localization and genic features that are distinct from steady-state eQTLs. Furthermore, the human orthologs associated with diet-responsive eQTLs are enriched for GWAS genes associated with human metabolic traits, suggesting that context-responsive eQTLs with more complex regulatory effects are likely to explain GWAS hits that do not seem to overlap with standard eQTLs. Our results highlight the complexity of genetic regulatory effects and the potential of eQTLs with disease-relevant GxE interactions in enhancing the understanding of GWAS signals for human complex disease using non-human primate models.

Intracellular Islatravir-Triphosphate in Dried Blood Spots and Peripheral Blood Mononuclear Cells from Pig-Tailed Macaques.

The purpose of this study was to evaluate the relationship between intracellular islatravir-triphosphate (ISL-TP) in paired peripheral blood mononuclear cells (PBMCs) and dried blood spots (DBS). Three pig-tailed macaques (PMs) were dosed with a single intravaginal extended-release ISL-etonogestrel film for a period of 31 days. After extraction and quantification, repeated measures correlation (rrm) was assessed between log-transformed DBS and PBMC ISL-TP concentrations. Twenty-six paired PBMC/DBS samples were included. Peak ISL-TP concentrations in DBS ranged from 262 to 913 fmol/punches, PBMC Cmax ranged from 427 to 857 fmol/106 cells. Repeated measures correlation yielded an rrm value of 0.96 (95% confidence interval 0.92-0.98; p < .0001). Importantly, ISL-TP was quantifiable in DBS and its pharmacokinetics were similar to PBMC in PMs. Human studies should evaluate DBS applications in clinical pharmacokinetic studies to help define ISL's place in the antiretroviral drug armamentarium.

Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques.

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or ß-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.

Development and validation of an LC-MS/MS based quantitative assay for marmoset insulin in serum.

Insulin is a peptide hormone that is secreted by the ß cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). Marmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin. Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled (13C, 15N) synthetic marmoset insulin tryptic peptide was used as internal standard. The assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 15.49 pmol/L and the limit of quantitation was 140.78 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L. In summary, this paper presents a simple, reproducible method to measure marmoset insulin in serum using LC-MS/MS.

Aromatase Inhibition Eliminates Sexual Receptivity Without Enhancing Weight Gain in Ovariectomized Marmoset Monkeys.

Context: Ovarian estradiol supports female sexual behavior and metabolic function. While ovariectomy (OVX) in rodents abolishes sexual behavior and enables obesity, OVX in nonhuman primates decreases, but does not abolish, sexual behavior, and inconsistently alters weight gain. Objective: We hypothesize that extra-ovarian estradiol provides key support for both functions, and to test this idea, we employed aromatase inhibition to eliminate extra-ovarian estradiol biosynthesis and diet-induced obesity to enhance weight gain. Methods: Thirteen adult female marmosets were OVX and received (1) estradiol-containing capsules and daily oral treatments of vehicle (E2; n = 5); empty capsules and daily oral treatments of either (2) vehicle (VEH, 1 mL/kg, n = 4), or (3) letrozole (LET, 1 mg/kg, n = 4). Results: After 7 months, we observed robust sexual receptivity in E2, intermediate frequencies in VEH, and virtually none in LET females (P = .04). By contrast, few rejections of male mounts were observed in E2, intermediate frequencies in VEH, and high frequencies in LET females (P = .04). Receptive head turns were consistently observed in E2, but not in VEH and LET females. LET females, alone, exhibited robust aggressive rejection of males. VEH and LET females demonstrated increased % body weight gain (P = .01). Relative estradiol levels in peripheral serum were E2 >>> VEH > LET, while those in hypothalamus ranked E2 = VEH > LET, confirming inhibition of local hypothalamic estradiol synthesis by letrozole. Conclusion: Our findings provide the first evidence for extra-ovarian estradiol contributing to female sexual behavior in a nonhuman primate, and prompt speculation that extra-ovarian estradiol, and in particular neuroestrogens, may similarly regulate sexual motivation in other primates, including humans.

Dopamine D3 receptor ligand suppresses the expression of levodopa-induced dyskinesia in nonhuman primate model of parkinson's disease.

Parkinson's disease (PD) is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. The incidence of PD increases with aging and the expected burden will continue to escalate with our aging population. Since its discovery in the 1961 levodopa has remained the gold standard pharmacotherapy for PD. However, the progressive nature of the neurodegenerative process in and beyond the nigrostriatal system causes a multitude of side effects, including levodopa-induced dyskinesia within 5 years of therapy. Attenuating dyskinesia has been a significant challenge in the clinical management of PD. We report on a small molecule that eliminates the expression of levodopa-induced dyskinesia and significantly improves PD-like symptoms. The lead compound PD13R we discovered is a dopamine D3 receptor partial agonist with high affinity and selectivity, orally active and with desirable drug-like properties. Future studies are aimed at developing this lead compound for treating PD patients with dyskinesia.

Validation of the Performance of A1HPV6, a Triage Blood Test for the Early Diagnosis and Prognosis of SARS-CoV-2 Infection.

BACKGROUND AND AIMS: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. METHODS: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and sex-adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. RESULTS: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97-0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99-0.99; P = .80), like for sensitivity 92% (85-96) vs 94% (88-97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. CONCLUSION: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

The Potential Role of Nonhuman Primate Models to Better Comprehend Early Life Immunity and Maternal Antibody Transfer.

Early life immunity is a complex field of research and there are still gaps in knowledge regarding the detailed mechanism of maternal antibody transfer, the impact of maternal antibodies on infant vaccine responses and the ontogeny of human early life immunity. A comprehensive understanding is necessary to identify requirements for early life vaccines and to improve early childhood immunization. New immunological methods have facilitated performing research in the youngest, however, some questions can only be addressed in animal models. To date, mostly murine models are used to study neonatal and infant immunity since they are well-described, easy to use and cost effective. Given their limitations especially in the transfer biology of maternal antibodies and the lack of infectivity of numerous human pathogens, this opinion piece discusses the potential and prerequisites of the nonhuman primate model in studying early life immunity and maternal antibody transfer.

Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys.

Although CRISPR/Cas9-mediated gene editing is widely applied to mimic human disorders, whether acute manipulation of disease-causing genes in the brain leads to behavioral abnormalities in non-human primates remains to be determined. Here we induced genetic mutations in MECP2, a critical gene linked to Rett syndrome (RTT) and autism spectrum disorders (ASD), in the hippocampus (DG and CA1-4) of adolescent rhesus monkeys (Macaca mulatta) in vivo via adeno-associated virus (AAV)-delivered Staphylococcus aureus Cas9 with small guide RNAs (sgRNAs) targeting MECP2. In comparison to monkeys injected with AAV-SaCas9 alone (n = 4), numerous autistic-like behavioral abnormalities were identified in the AAV-SaCas9-sgMECP2-injected monkeys (n = 7), including social interaction deficits, abnormal sleep patterns, insensitivity to aversive stimuli, abnormal hand motions, and defective social reward behaviors. Furthermore, some aspects of ASD and RTT, such as stereotypic behaviors, did not appear in the MECP2 gene-edited monkeys, suggesting that different brain areas likely contribute to distinct ASD symptoms. This study showed that acute manipulation of disease-causing genes via in vivo gene editing directly led to behavioral changes in adolescent primates, paving the way for the rapid generation of genetically engineered non-human primate models for neurobiological studies and therapeutic development.

Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina.

Retinitis pigmentosa (RP) is a form of inherited retinal degenerative diseases that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.

Structural development of cortical lobes during the first 6 months of life in infant macaques.

This study mapped the developmental trajectories of cortical regions in comparison to overall brain growth in typically developing, socially-housed infant macaques. Volumetric changes of cortical brain regions were examined longitudinally between 2-24 weeks of age (equivalent to the first 2 years in humans) in 21 male rhesus macaques. Growth of the prefrontal, frontal, parietal, occipital, and temporal cortices (visual and auditory) was examined using MRI and age-specific infant macaque brain atlases developed by our group. Results indicate that cortical volumetric development follows a cubic growth curve, but maturational timelines and growth rates are region-specific. Total intracranial volume (ICV) increased significantly during the first 5 months of life, leveling off thereafter. Prefrontal and temporal visual cortices showed fast volume increases during the first 16 weeks, followed by a plateau, and significant growth again between 20-24 weeks. Volume of the frontal and temporal auditory cortices increased substantially between 2-24 weeks. The parietal cortex showed a significant volume increase during the first 4 months, whereas the volume of the occipital lobe increased between 2-12 weeks and plateaued thereafter. These developmental trajectories show similarities to cortical growth in human infants, providing foundational information necessary to build nonhuman primate (NHP) models of human neurodevelopmental disorders.

Lack of Evidence for Microbiota in the Placental and Fetal Tissues of Rhesus Macaques.

The prevailing paradigm in obstetrics has been the sterile womb hypothesis. However, some are asserting that the placenta, intra-amniotic environment, and fetus harbor microbial communities. The objective of this study was to determine whether the fetal and placental tissues of rhesus macaques harbor bacterial communities. Fetal, placental, and uterine wall samples were obtained from cesarean deliveries without labor (∼130/166 days gestation). The presence of bacteria in the fetal intestine and placenta was investigated through culture. The bacterial burden and profiles of the placenta, umbilical cord, and fetal brain, heart, liver, and colon were determined through quantitative real-time PCR and DNA sequencing. These data were compared with those of the uterine wall as well as to negative and positive technical controls. Bacterial cultures of fetal and placental tissues yielded only a single colony of Cutibacterium acnes This bacterium was detected at a low relative abundance (0.02%) in the 16S rRNA gene profile of the villous tree sample from which it was cultured, yet it was also identified in 12/29 background technical controls. The bacterial burden and profiles of fetal and placental tissues did not exceed or differ from those of background technical controls. By contrast, the bacterial burden and profiles of positive controls exceeded and differed from those of background controls. Among the macaque samples, distinct microbial signals were limited to the uterine wall. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the fetal and placental tissues of rhesus macaques.IMPORTANCE Microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) has been causally linked to pregnancy complications, especially preterm birth. Therefore, if the placenta and the fetus are typically populated by low-biomass microbial communities, current understanding of the role of microbes in reproduction and pregnancy outcomes will need to be fundamentally reconsidered. Could these communities be of benefit by competitively excluding potential pathogens or priming the fetal immune system for the microbial bombardment it will experience upon delivery? If so, what properties (e.g., microbial load and community membership) of these microbial communities preclude versus promote intra-amniotic infection? Given the ramifications of the in utero colonization hypothesis, critical evaluation is required. In this study, using multiple modes of microbiologic inquiry (i.e., culture, quantitative real-time PCR [qPCR], and DNA sequencing) and controlling for potential background DNA contamination, we did not find consistent evidence for microbial communities in the placental and fetal tissues of rhesus macaques.