Considerations for the Use of Viral Vectors in Nonhuman Primates

Vectorology for Optogenetics and Chemogenetics

2023 Feb 07

Bohlen, M;Tremblay, S;
| DOI: 10.1007/978-1-0716-2918-5_16

The development of new genetic tools has revolutionized our ability to study the functional role of specific neuronal populations and circuits generating behavior. Although this revolution has already taken place in small animal models such as mice, adoption of these techniques has been relatively slow for animals more closely related to humans, such as nonhuman primates. Current challenges include effective delivery to much larger structural targets in the primate brain, cell-type specific transduction, and immunological responses. In this chapter, we will review some of the challenges and considerations that are specific to using these viral technologies in the nonhuman primate brain. Ultimately, these challenges can be met with new advances in surgical technique and gene therapy that will spin out new viral vectors with enhanced features able to compensate for the limitations of current vectors. As the existing challenges are circumvented, this will lead to a revolution in primate neuroscientific research and a greater understanding of the functional role circuits play in complex behaviors relevant to human neurological and psychiatric diseases.

HIV integration in the human brain is linked to microglial activation and 3D genome remodeling

Molecular cell

2022 Dec 15

Plaza-Jennings, AL;Valada, A;O'Shea, C;Iskhakova, M;Hu, B;Javidfar, B;Ben Hutta, G;Lambert, TY;Murray, J;Kassim, B;Chandrasekaran, S;Chen, BK;Morgello, S;Won, H;Akbarian, S;
PMID: 36525955 | DOI: 10.1016/j.molcel.2022.11.016

To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.

Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis

Cell reports

2022 Nov 22

Bakker, W;Imbernon, M;Salinas, CG;Moro Chao, DH;Hassouna, R;Morel, C;Martin, C;Leger, C;Denis, RGP;Castel, J;Peter, A;Heni, M;Maetzler, W;Nielsen, HS;Duquenne, M;Schwaninger, M;Lundh, S;Johan Hogendorf, WF;Gangarossa, G;Secher, A;Hecksher-Sørensen, J;Pedersen, TÅ;Prevot, V;Luquet, S;
PMID: 36417883 | DOI: 10.1016/j.celrep.2022.111698

Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treatment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of insulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanistically, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selectively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related signals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.

TGFβ signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus

Developmental biology

2022 May 26

Clayton, SW;Angermeier, A;Halbrooks, JE;McCardell, R;Serra, R;
PMID: 35644252 | DOI: 10.1016/j.ydbio.2022.05.013

We previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFβRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFβ signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment

Scientific reports

2022 Mar 08

Scheff, NN;Wall, IM;Nicholson, S;Williams, H;Chen, E;Tu, NH;Dolan, JC;Liu, CZ;Janal, MN;Bunnett, NW;Schmidt, BL;
PMID: 35260737 | DOI: 10.1038/s41598-022-08005-6

Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

NMNAT2 is downregulated in glaucomatous RGCs, and RGC-specific gene therapy rescues neurodegeneration and visual function

Molecular therapy : the journal of the American Society of Gene Therapy

2022 Jan 31

Fang, F;Zhuang, P;Feng, X;Liu, P;Liu, D;Huang, H;Li, L;Chen, W;Liu, L;Sun, Y;Jiang, H;Ye, J;Hu, Y;
PMID: 35114390 | DOI: 10.1016/j.ymthe.2022.01.035

The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse γ-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline, and that modulating RGC-intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

Spatial expression of IKK-alpha is associated with a differential mutational landscape and survival in primary colorectal cancer

British journal of cancer

2022 Feb 16

Patel, M;Pennel, KAF;Quinn, JA;Hood, H;Chang, DK;Biankin, AV;Rebus, S;Roseweir, AK;Park, JH;Horgan, PG;McMillan, DC;Edwards, J;
PMID: 35173303 | DOI: 10.1038/s41416-022-01729-2

To understand the relationship between key non-canonical NF-κB kinase IKK-alpha(α), tumour mutational profile and survival in primary colorectal cancer.Immunohistochemical expression of IKKα was assessed in a cohort of 1030 patients who had undergone surgery for colorectal cancer using immunohistochemistry. Mutational tumour profile was examined using a customised gene panel. Immunofluorescence was used to identify the cellular location of punctate IKKα expression.Two patterns of IKKα expression were observed; firstly, in the tumour cell cytoplasm and secondly as discrete 'punctate' areas in a juxtanuclear position. Although cytoplasmic expression of IKKα was not associated with survival, high 'punctate' IKKα expression was associated with significantly reduced cancer-specific survival on multivariate analysis. High punctate expression of IKKα was associated with mutations in KRAS and PDGFRA. Dual immunofluorescence suggested punctate IKKα expression was co-located with the Golgi apparatus.These results suggest the spatial expression of IKKα is a potential biomarker in colorectal cancer. This is associated with a differential mutational profile highlighting possible distinct signalling roles for IKKα in the context of colorectal cancer as well as potential implications for future treatment strategies using IKKα inhibitors.

Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease

Brain

2021 Sep 30

Steinkellner, T;Conrad, WS;Kovacs, I;Rissman, RA;
| DOI: 10.1093/brain/awab373

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson’s disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving DA neurons from postmortem Parkinson’s disease subjects. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson’s disease.

Scientific Evidence Supporting Coronavirus Disease 2019 (COVID-19) Vaccine Efficacy and Safety in People Planning to Conceive or Who Are Pregnant or Lactating

Obstetrics and gynecology

2021 Nov 02

Girardi, G;Bremer, AA;
PMID: 34727554 | DOI: 10.1097/AOG.0000000000004636

Three coronavirus disease 2019 (COVID-19) vaccines have been authorized for use in the United States; specifically, the Pfizer-BioNTech, Moderna, and Johnson & Johnson-Janssen COVID-19 vaccines were granted emergency use authorization by the U.S. Food and Drug Administration in late 2020 and early 2021. Vaccination coverage and intent among adults are lowest among those aged 18-39 years and among females in particular. In females of reproductive age, enthusiasm for receiving a COVID-19 vaccine may be negatively affected by claims currently circulating widely on diverse social media platforms regarding the vaccines adversely affecting fertility and pregnancy. Yet it is important to note that these claims are anecdotal in nature and not supported by the available scientific evidence. It is also imperative that the effects of COVID-19 vaccine on reproductive health are clarified. Herein, we discuss the existing scientific data supporting COVID-19 vaccine safety and efficacy in people who are planning to conceive or who are pregnant or lactating and highlight the importance of COVID-19 vaccination in females of reproductive age.

Quantitative Off-Target Detection of Epstein-Barr Virus-Derived DNA in Routine Molecular Profiling of Hematopoietic Neoplasms by Panel-Based Hybrid-Capture Next-Generation Sequencing

The Journal of molecular diagnostics : JMD

2021 Nov 18

Petrova-Drus, K;Quesada, AE;Bowman, AS;Ptashkin, R;Yao, J;Arcila, ME;Ho, C;Moung, C;Regalado, J;Benayed, R;Benhamida, JK;Galera, PK;Dogan, A;Vanderbilt, C;
PMID: 34801704 | DOI: 10.1016/j.jmoldx.2021.10.009

Epstein-Barr virus (EBV) is associated with hematologic and solid tumors. In this study, we utilized a hybridization capture-based next-generation sequencing (NGS) platform that targets 400 genes associated with hematological malignancies to detect and quantify nontargeted viral-derived EBV reads that aligned to the EBV reference contig (NC_007605). We evaluated 5234 samples from 3636 unique patients with hematological neoplasms and found that 100 samples (1.9%) in 93 unique patients had ≥6 EBV reads (range, 6 to 32,325; mean, 827.5; median, 54). Most (n = 73, 73%) represented known EBV-associated conditions, and the most common was post-transplant lymphoproliferative disorders (n = 21, 29%). Documented EBV viremia accounted for a moderate quantity of EBV reads in 4 of 27 samples corresponding to conditions not known to be EBV associated, whereas suspected viremia or low-level activation was likely the etiology in the remaining 23 samples. A good correlation (Spearman r = 0.8; 95% CI, 0.74-0.85) was found between EBV reads by NGS and systematic semiquantitative EBER in situ hybridization assessment in 162 available samples, particularly at higher level of EBV involvement. An optimal threshold for significant morphologic EBV involvement was found to be ≥10 reads by the receiver operating characteristic analysis (area under the curve, 0.990; 95% CI, 0.9974%-1.000%). Thus, in addition to mutational analysis, hybrid-capture-based NGS panels can be used to detect and quantitate off-target EBV-derived viral DNA, which correlates well with morphology.

Molecular mechanisms in IL-1β-mediated decorin production by decidual cells

Molecular human reproduction

2021 Nov 27

Halari, CD;Renaud, SJ;Lala, PK;
PMID: 34915564 | DOI: 10.1093/molehr/gaab068

Decorin, a small leucine-rich proteoglycan produced by decidual cells restrains trophoblast differentiation, migration and invasiveness of extra-villous trophoblast cells. Decidual overproduction of decorin is associated with preeclampsia, and elevated decorin levels in maternal plasma are a predictive biomarker of preeclampsia. Furthermore, decorin plays an autocrine role in maturation of human endometrial stromal cells into decidual cells. Thus, a balanced decorin production by the decidua is critical for healthy pregnancy. However, the molecular mechanisms regulating decorin production by the decidua are unclear. Interleukin-1 beta is an inflammation-associated multi-functional cytokine, and is reported to induce decidualization in primates. Hence, the present study was designed: (i) to test if exogenous Interleukin-1 beta stimulated decorin production by human endometrial stromal cells; and if so, (ii) to identify the cellular source of Interleukin-1 beta in first trimester decidual tissue; (iii) to identify the downstream molecular partners in Interleukin-1 beta mediated decorin production by human endometrial stromal cells. Results revealed that (i) amongst multiple pro-inflammatory cytokines tested, Interleukin-1 beta alone stimulated decorin production by these cells; (ii) both macrophages and decidual cells in first trimester decidua produced Interleukin-1 beta; (iii) Interleukin-1 beta mediated decorin production was dependent on Interleukin-1 receptor activation, followed by activation and nuclear translocation of nuclear factor kappa B and its binding to the decorin promoter. These results reveal that Interleukin-1 beta plays a novel role in inducing decorin production by human endometrial stromal cells by activating nuclear factor kappa B.

Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry

Nature communications

2021 Oct 08

van Maldegem, F;Valand, K;Cole, M;Patel, H;Angelova, M;Rana, S;Colliver, E;Enfield, K;Bah, N;Kelly, G;Tsang, VSK;Mugarza, E;Moore, C;Hobson, P;Levi, D;Molina-Arcas, M;Swanton, C;Downward, J;
PMID: 34625563 | DOI: 10.1038/s41467-021-26214-x

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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