Lipid nanoparticle-mediated mRNA delivery to CD34 cells in rhesus monkeys.

Transplantation of ex vivo engineered hematopoietic stem cells (HSCs) can lead to robust clinical responses but carries risks of adverse events from bone marrow mobilization, chemotherapy conditioning and other factors. HSCs have been modified in vivo using lipid nanoparticles (LNPs) decorated with targeting moieties, which increases manufacturing complexity. Here we screen 105 LNPs without targeting ligands for effective homing to the bone marrow in mouse.

Systematic multi-trait AAV capsid engineering for efficient gene delivery.

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids.

AAV5 Delivery of CRISPR/Cas9 Mediates Genome Editing in the Lungs of Young Rhesus Monkeys.

Genome editing has the potential to treat genetic diseases in a variety of tissues, including the lung. We have previously developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing in the airways of mice. To validate this delivery vehicle in a large animal model, we have shown that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys.

Engineered IgM and IgG cleaving enzymes for mitigating antibody neutralization and complement activation in AAV gene transfer.

Systemic dosing of adeno-associated viral (AAV) vectors poses potential risk of adverse side effects including complement activation triggered by anti-capsid immunity. Due to the multifactorial nature of toxicities observed in this setting, a wide spectrum of immune modulatory regimens are being investigated in the clinic. Here, we discover an IgM cleaving enzyme (IceM) that degrades human IgM, a key trigger in the anti-AAV immune cascade.

Expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF in the developing rhesus monkey retina.

The fovea centralis (fovea) is a specialized region of the primate retina that plays crucial roles in high-resolution visual acuity and color perception. The fovea is characterized by a high density of cone photoreceptors and no rods, and unique anatomical properties that contribute to its remarkable visual capabilities. Early histological analyses identified some of the key events that contribute to foveal development, but the mechanisms that direct the specification of this area are not understood.

Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP.

Mathematical Modeling of Rhesus Cytomegalovirus Transplacental Transmission in Seronegative Rhesus Macaques.

Approximately 0.7% of infants are born with congenital cytomegalovirus (CMV), making it the most common congenital infection. About 1 in 5 congenitally infected babies will suffer long-term sequelae, including sensorineural deafness, intellectual disability, and epilepsy.

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques.

Unlabelled: Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV.

Encapsulated Allografts Preclude Host Sensitization and Promote Ovarian Endocrine Function in Ovariectomized Young Rhesus Monkeys and Sensitized Mice.

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immunoisolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naïve ovariectomized BALB/c mice responded to the circulating gonadotropins and maintained function for 4 months, as evident by regular estrous cycles and the presence of antral follicles in the retrieved grafts.

Shuttle Peptide Delivers Base Editor RNPs to Rhesus Monkey Airway Epithelial Cells .

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, to improve base editor RNP delivery, we optimized S10 to derive the S315 peptide.

Rapid molecular imaging of active thrombi using aptamer-antidote probes.

Pathological blood clotting, or thrombosis, limits vital blood flow to organs; such deprivation can lead to catastrophic events including myocardial infarction, pulmonary embolism, and ischemic stroke. Prompt restoration of blood flow greatly improves outcomes. We explored whether aptamers could serve as molecular imaging probes to rapidly detect thrombi.

Late gene expression-deficient cytomegalovirus vectors elicit conventional T cells that do not protect against SIV.

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71).

Imaging the fetal nonhuman primate brain with SV2A positron emission tomography (PET).

Purpose: Exploring synaptic density changes during brain growth is crucial to understanding brain development. Previous studies in nonhuman primates report a rapid increase in synapse number between the late gestational period and the early neonatal period, such that synaptic density approaches adult levels by birth. Prenatal synaptic development may have an enduring impact on postnatal brain development, but precisely how synaptic density changes in utero are unknown because current methods to quantify synaptic density are invasive and require post-mortem brain tissue.

Translational Utility of the Nonhuman Primate Model.

Nonhuman primates are essential for the study of human disease and to explore the safety of new diagnostics and therapies proposed for human use. They share similar genetic, physiologic, immunologic, reproductive, and developmental features with humans and thus have proven crucial for the study of embryonic/fetal development, organ system ontogeny, and the role of the maternal-placental-fetal interface in health and disease. The fetus may be exposed to a variety of inflammatory stimuli including infectious microbes as well as maternal inflammation, which can result from infections, obesity, or environmental exposures.

Nonhuman Primates in Translational Research.

Nonhuman primates are critically important animal models in which to study complex human diseases, understand biological functions, and address the safety of new diagnostics and therapies proposed for human use. They have genetic, physiologic, immunologic, and developmental similarities when compared to humans and therefore provide important preclinical models of human health and disease. This review highlights select research areas that demonstrate the importance of nonhuman primates in translational research.

An Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection.

Greater Number of Microglia in Telencephalic Proliferative Zones of Human and Nonhuman Primate Compared with Other Vertebrate Species.

Microglial cells, the innate immune cells of the brain, are derived from yolk sac precursor cells, begin to colonize the telencephalon at the onset of cortical neurogenesis, and occupy specific layers including the telencephalic proliferative zones. Microglia are an intrinsic component of cortical germinal zones, establish extensive contacts with neural precursor cells (NPCs) and developing cortical vessels, and regulate the size of the NPC pool through mechanisms that include phagocytosis. Microglia exhibit notable differences in number and distribution in the prenatal neocortex between rat and old world nonhuman primate telencephalon, suggesting that microglia possess distinct properties across vertebrate species.

Early Embryonic Loss Following Intravaginal Zika Virus Challenge in Rhesus Macaques.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) and is primarily transmitted by species mosquitoes; however, ZIKV can also be sexually transmitted. During the initial epidemic and in places where ZIKV is now considered endemic, it is difficult to disentangle the risks and contributions of sexual versus vector-borne transmission to adverse pregnancy outcomes. To examine the potential impact of sexual transmission of ZIKV on pregnancy outcome, we challenged three rhesus macaques () three times intravaginally with 1 x 10 PFU of a low passage, African lineage ZIKV isolate (ZIKV-DAK) in the first trimester (~30 days gestational age).

Gut germinal center regeneration and enhanced antiviral immunity by mesenchymal stem/stromal cells in SIV infection.

Although antiretroviral therapy suppresses HIV replication, it does not eliminate viral reservoirs or restore damaged lymphoid tissue, posing obstacles to HIV eradication. Using the SIV model of AIDS, we investigated the effect of mesenchymal stem/stromal cell (MSC) infusions on gut mucosal recovery, antiviral immunity, and viral suppression and determined associated molecular/metabolic signatures. MSC administration to SIV-infected macaques resulted in viral reduction and heightened virus-specific responses.

MicroRNA Signatures of the Developing Primate Fovea.

Rod and cone photoreceptors differ in their shape, photopigment expression, synaptic connection patterns, light sensitivity, and distribution across the retina. Although rods greatly outnumber cones, human vision is mostly dependent on cone photoreceptors since cones are essential for our sharp visual acuity and color discrimination. In humans and other primates, the (fovea), a specialized region of the central retina, contains the highest density of cones.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters.

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular).