Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene

Nature communications

2023 Jun 06

Vansandt, LM;Meinsohn, MC;Godin, P;Nagykery, N;Sicher, N;Kano, M;Kashiwagi, A;Chauvin, M;Saatcioglu, HD;Barnes, JL;Miller, AG;Thompson, AK;Bateman, HL;Donelan, EM;González, R;Newsom, J;Gao, G;Donahoe, PK;Wang, D;Swanson, WF;Pépin, D;
PMID: 37280258 | DOI: 10.1038/s41467-023-38721-0

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Spatiotemporal transcriptome analysis reveals critical roles for mechano-sensing genes at the border zone in remodeling after myocardial infarction

Nature Cardiovascular Research

2022 Nov 17

Yamada, S;Ko, T;Hatsuse, S;Nomura, S;Zhang, B;Dai, Z;Inoue, S;Kubota, M;Sawami, K;Yamada, T;Sassa, T;Katagiri, M;Fujita, K;Katoh, M;Ito, M;Harada, M;Toko, H;Takeda, N;Morita, H;Aburatani, H;Komuro, I;
| DOI: 10.1038/s44161-022-00140-7

The underlying mechanisms of ventricular remodeling after myocardial infarction (MI) remain largely unknown. In this study, we performed an integrative analysis of spatial transcriptomics and single-nucleus RNA sequencing (snRNA-seq) in a murine MI model and found that mechanical stress-response genes are expressed at the border zone and play a critical role in left ventricular remodeling after MI. An integrative analysis of snRNA-seq and spatial transcriptome of the heart tissue after MI identified the unique cluster that appeared at the border zone in an early stage, highly expressing mechano-sensing genes, such as Csrp3. AAV9-mediated gene silencing and overexpression of Csrp3 demonstrated that upregulation of Csrp3 plays critical roles in preventing cardiac remodeling after MI by regulation of genes associated with mechano-sensing. Overall, our study not only provides an insight into spatiotemporal molecular changes after MI but also highlights that the mechano-sensing genes at the border zone act as adaptive regulators of left ventricular remodeling.

Preclinical evaluation of ADVM-062, a novel intravitreal gene therapy vector for the treatment of blue cone monochromacy

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

2023 Mar 16

Hanna, K;Nieves, J;Dowd, C;Bender, KO;Sharma, P;Singh, B;Renz, M;Ver Hoeve, JN;Cepeda, D;Gelfman, CM;Riley, BE;Grishanin, RN;
PMID: 36932675 | DOI: 10.1016/j.ymthe.2023.03.011

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.

Molecular profiling of enteric nervous system cell lineages

Nature protocols

2022 Jun 08

Obata, Y;Castaño, Á;Fallesen, TL;Bon-Frauches, AC;Boeing, S;Huseynova, A;McCallum, S;Lasrado, R;Heanue, TA;Pachnis, V;
PMID: 35676375 | DOI: 10.1038/s41596-022-00697-4

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo

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

2022 May 16

Gorsuch, CL;Nemec, P;Yu, M;Xu, S;Han, D;Smith, J;Lape, J;van Buuren, N;Ramirez, R;Muench, RC;Holdorf, MM;Feierbach, B;Falls, G;Holt, J;Shoop, W;Sevigny, E;Karriker, F;Brown, RV;Joshi, A;Goodwin, T;Tam, YK;Lin, PJC;Semple, SC;Leatherbury, N;Delaney Iv, WE;Jantz, D;Rhoden Smith, A;
PMID: 35581938 | DOI: 10.1016/j.ymthe.2022.05.013

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

CONSEQUENCES OF mTOR INHIBITION ON AAV HEPATIC TRANSDUCTION EFFICACY

Human gene therapy

2021 Sep 24

Perez-Iturralde, A;Carte, B;Aldabe, R;
PMID: 34555962 | DOI: 10.1089/hum.2021.171

The efficiency of recombinant Adeno-associated virus vectors (AAV) transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mTOR inhibitors.- rapamycin, MLN0128, RapaLink-1 -were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involve in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.

The implication of hinge 1 and hinge 4 in micro-dystrophin gene therapy for Duchenne muscular dystrophy

Human gene therapy

2022 Oct 31

Wasala, LP;Watkins, T;Wasala, N;Burke, M;Yue, Y;Lai, Y;Yao, G;Duan, D;
PMID: 36310439 | DOI: 10.1089/hum.2022.180

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins are currently in clinical trials. They all carry H1 and H4. Here we investigated whether these two hinges are essential for micro-dystrophin function in murine DMD models. Three otherwise identical micro-dystrophins were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ∆H1 (without H1), and ∆H4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent micro-dystrophin expression in total muscle lysate. However, only H1/H4 and ∆H1 showed correct sarcolemmal localization. ∆H4 mainly existed as subsarcolemmal aggregates. H1/H4 and ∆H1, but not ∆H4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ∆H1, but not by ∆H4. Next, we compared H1/H4 and ∆H1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months post-injection, H1/H4 significantly outperformed ∆H1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable ECG improvements. We conclude that H4 is essential for micro-dystrophin function and H1 facilitates force production. Our findings will help develop next-generation micro-dystrophin gene therapy.

Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

Cytotherapy

2022 May 12

Giardino Torchia, ML;Letizia, M;Gilbreth, R;Merlino, A;Sult, E;Monks, N;Chesebrough, J;Tammali, R;Chu, N;Tong, J;Meekin, J;Schifferli, K;Vashisht, K;DaCosta, K;Clarke, L;Gesse, C;Yao, XT;Bridges, C;Moody, G;
PMID: 35570170 | DOI: 10.1016/j.jcyt.2022.03.008

Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.

CNS-dominant human FMRP isoform rescues seizures, fear, and sleep abnormalities in Fmr1-KO mice

JCI insight

2023 Jun 08

Wong, H;Hooper, AW;Kang, HR;Lee, SJ;Zhao, J;Sadhu, C;Rawat, S;Gray, SJ;Hampson, DR;
PMID: 37288657 | DOI: 10.1172/jci.insight.169650

Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.

An Overview of Gene Editing Modalities and Related Non-clinical Testing Considerations

International journal of toxicology

2023 Feb 10

Vats, S;Ballesteros, C;Hung, S;Sparapani, S;Wong, K;Haruna, J;Li, C;Authier, S;
PMID: 36762691 | DOI: 10.1177/10915818231153996

Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.

Incisionless targeted adeno-associated viral vector delivery to the brain by focused ultrasound-mediated intranasal administration

EBioMedicine

2022 Sep 21

Ye, D;Yuan, J;Yang, Y;Yue, Y;Hu, Z;Fadera, S;Chen, H;
PMID: 36152518 | DOI: 10.1016/j.ebiom.2022.104277

Adeno-associated viral (AAV) vectors are currently the leading platform for gene therapy with the potential to treat a variety of central nervous system (CNS) diseases. There are numerous methods for delivering AAVs to the CNS, such as direct intracranial injection (DI), intranasal delivery (IN), and intravenous injection with focused ultrasound-induced blood-brain barrier disruption (FUS-BBBD). However, non-invasive and efficient delivery of AAVs to the brain with minimal systemic toxicity remain the major challenge. This study aims to investigate the potential of focused ultrasound-mediated intranasal delivery (FUSIN) in AAV delivery to brain.Mice were intranasally administered with AAV5 encoding enhanced green fluorescence protein (AAV5-EGFP) followed by FUS sonication in the presence of systemically injected microbubbles. Mouse brains and other major organs were harvested for immunohistological staining, PCR quantification, and in situ hybridization. The AAV delivery outcomes were compared with those of DI, FUS-BBBD, and IN delivery.FUSIN achieved safe and efficient delivery of AAV5-EGFP to spatially targeted brain locations, including a superficial brain site (cortex) and a deep brain region (brainstem). FUSIN achieved comparable delivery outcomes as the established DI, and displayed 414.9-fold and 2073.7-fold higher delivery efficiency than FUS-BBBD and IN. FUSIN was associated with minimal biodistribution in peripheral organs, which was comparable to that of DI.Our results suggest that FUSIN is a promising technique for non-invasive, efficient, safe, and spatially targeted AAV delivery to the brain.National Institutes of Health (NIH) grants R01EB027223, R01EB030102, R01MH116981, and UG3MH126861.

Probing regenerative heterogeneity of corticospinal neurons with scRNA-Seq

Research square

2023 Feb 21

Kim, H;Saikia, J;Monte, K;Ha, E;Romaus-Sanjurjo, D;Sanchez, J;Moore, A;Hernaiz-Llorens, M;Chavez-Martinez, C;Agba, C;Li, H;Lusk, D;Cervantes, K;Zheng, B;
PMID: 36865182 | DOI: 10.21203/rs.3.rs-2588274/v1

The corticospinal tract (CST) is clinically important for the recovery of motor functions after spinal cord injury. Despite substantial progress in understanding the biology of axon regeneration in the central nervous system (CNS), our ability to promote CST regeneration remains limited. Even with molecular interventions, only a small proportion of CST axons regenerate1. Here we investigate this heterogeneity in the regenerative ability of corticospinal neurons following PTEN and SOCS3 deletion with patch-based single cell RNA sequencing (scRNA-Seq)2,3, which enables deep sequencing of rare regenerating neurons. Bioinformatic analyses highlighted the importance of antioxidant response and mitochondrial biogenesis along with protein translation. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Applying Garnett4, a supervised classification method, to our dataset gave rise to a Regenerating Classifier (RC), which, when applied to published scRNA-Seq data, generates cell type- and developmental stage-appropriate classifications. While embryonic brain, adult dorsal root ganglion and serotonergic neurons are classified as Regenerators, most neurons from adult brain and spinal cord are classified as Non-regenerators. Adult CNS neurons partially revert to a regenerative state soon after injury, which is accelerated by molecular interventions. Our data indicate the existence of universal transcriptomic signatures underlying the regenerative abilities of vastly different neuronal populations, and further illustrate that deep sequencing of only hundreds of phenotypically identified CST neurons has the power to reveal new insights into their regenerative biology.

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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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