Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens
Nature communications
2021 May 18
Walter, ND;Born, SEM;Robertson, GT;Reichlen, M;Dide-Agossou, C;Ektnitphong, VA;Rossmassler, K;Ramey, ME;Bauman, AA;Ozols, V;Bearrows, SC;Schoolnik, G;Dolganov, G;Garcia, B;Musisi, E;Worodria, W;Huang, L;Davis, JL;Nguyen, NV;Nguyen, HV;Nguyen, ATV;Phan, H;Wilusz, C;Podell, BK;Sanoussi, ND;de Jong, BC;Merle, CS;Affolabi, D;McIlleron, H;Garcia-Cremades, M;Maidji, E;Eshun-Wilson, F;Aguilar-Rodriguez, B;Karthikeyan, D;Mdluli, K;Bansbach, C;Lenaerts, AJ;Savic, RM;Nahid, P;Vásquez, JJ;Voskuil, MI;
PMID: 34006838 | DOI: 10.1038/s41467-021-22833-6
There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.
Exposure to microbial metabolite butyrate prolongs the survival time and changes the growth pattern of HPV16 E6/E7-immortalized keratinocytes in vivo
The American journal of pathology
2021 Jun 29
Li, M;McGhee, EM;Shinno, L;Lee, K;Lin, YL;
PMID: 34214507 | DOI: 10.1016/j.ajpath.2021.06.005
Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. While cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure potentially can increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. Our results suggest that tonsillar bacteria's fermentation products may play an important role in the long-term persistence of high risk-HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.
Adeno-associated viral vector serotype 9-based gene replacement therapy for SURF1-related Leigh syndrome
Molecular Therapy - Methods & Clinical Development
2021 Sep 01
Ling, Q;Rioux, M;Hu, Y;Lee, M;Gray, S;
| DOI: 10.1016/j.omtm.2021.09.001
SURF1 (surfeit locus protein 1)-related Leigh syndrome is an early-onset neurodegenerative disorder, characterized by reduction in complex IV activity, resulting in disrupted mitochondrial function. Currently, there are no treatment options available. To test our hypothesis that adeno-associated viral vector serotype 9 (AAV9)/human SURF1 (hSURF1) gene replacement therapy can provide a potentially meaningful and long-term therapeutic benefit, we conducted preclinical efficacy studies using SURF1 knockout mice and safety evaluations with wild-type (WT) mice. Our data indicate that with a single intrathecal (i.t.) administration, our treatment partially and significantly rescued complex IV activity in all tissues tested, including liver, brain, and muscle. Accordingly, complex IV content (examined via MT-CO1 protein expression level) also increased with our treatment. In a separate group of mice, AAV9/hSURF1 mitigated the blood lactic acidosis induced by exhaustive exercise at 9 months post-dosing. A toxicity study in WT mice showed no adverse effects in either the in-life portion or after microscopic examination of major tissues up to a year following the same treatment regimen. Taken together, our data suggest a single dose, i.t. administration of AAV9/hSURF1 is safe and effective in improving biochemical abnormalities induced by SURF1 deficiency with potential applicability for SURF1-related Leigh syndrome patients.
Molecular Therapy - Methods & Clinical Development
2021 Dec 01
Sihn, C;Handyside, B;Liu, S;Zhang, L;Murphy, R;Yates, B;Xie, L;Torres, R;Russell, C;O’Neill, C;Pungor, E;Bunting, S;Fong, S;
| DOI: 10.1016/j.omtm.2021.12.004
Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation Factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissue. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and nonhuman primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and nonhuman primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.
Molecular Therapy - Nucleic Acids
2022 Feb 01
Alsing, S;Doktor, T;Askou, A;Jensen, E;Ahmadov, U;Kristensen, L;Andresen, B;Aagaard, L;Corydon, T;
| DOI: 10.1016/j.omtn.2022.02.019
Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety we have generated VEGFA-targeting agshRNAs and miRNA-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi-effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared to their shRNA counterparts. RNAseq analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared to their agshRNA counterparts and activation of immune related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in RPE-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.
First-in-human DR5 PET reveals insufficient DR5 expression in patients with gastrointestinal cancer
Journal for immunotherapy of cancer
2021 Jul 01
Wang, S;Zhu, H;Li, Y;Ding, J;Wang, F;Ding, L;Wang, X;Zhao, J;Zhang, Y;Yao, Y;Zhou, T;Li, N;Wu, A;Yang, Z;
PMID: 34301815 | DOI: 10.1136/jitc-2021-002926
Death receptor 5 (DR5) is a promising therapeutic target for cancer therapy. However, many clinical trials of DR5 agonists failed to show significant therapeutic efficacy in patients with cancer. The study aimed to investigate the feasibility of using 89Zr-CTB006 positron emission tomography (PET) for noninvasive imaging of DR5 expression in preclinical models and patients with gastrointestinal (GI) cancers.Balb/c, Sp2/0 xenograft and patient-derived tumor xenograft were employed for micro-PET/CT imaging in vivo. In the clinical study, patients with GI cancers planning to undergo surgical operation were enrolled and underwent 18F-FDG and 89Zr-CTB006 PET/CT. The tumor tissues were obtained through surgical operation and DR5 expression levels were confirmed by RNAscope.Preclinical studies showed that 89Zr-CTB006 PET could specifically detect DR5 expression levels in vivo. Twenty-one patients, including nine gastric cancers and 12 colorectal cancers, were enrolled. The biodistribution showed high uptake in the liver and spleen and low uptake in the brain, lung and muscle with an acceptable whole-body dosimetry of 0.349 mSv/MBq. Strikingly, the adrenal glands maintained stable high uptake over the entire examination in all patients. The tumor lesions showed different levels of uptake of 89Zr-CTB006 with a mean maximum standardized uptake value (SUVmax) of 6.63±3.29 (range 1.8-13.8). Tumor tissue was obtained from 18 patients, and 89Zr-CTB006 uptake in patients with RNAscope scores of 3-4 was significantly higher than that in patients with scores of 0-2. An SUVmax of 9.3 at 48 hours and 6.3 at 72 hours could be used to discriminate the DR5 expression status of tumors both with a sensitivity and specificity of 100% and 92.9%, respectively.89Zr-CTB006 PET/CT is capable of detecting DR5 expression in cancer patients and is a promising approach to screen patients with DR5 overexpression.
Shisa6 mediates cell-type specific regulation of depression in the nucleus accumbens
Molecular psychiatry
2021 Jul 12
Kim, HD;Wei, J;Call, T;Quintus, NT;Summers, AJ;Carotenuto, S;Johnson, R;Ma, X;Xu, C;Park, JG;Qiu, S;Ferguson, D;
PMID: 34253865 | DOI: 10.1038/s41380-021-01217-8
Depression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.
Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl-/- Mice
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
2021 Jun 02
Kinosh*ta, Y;Mohamed, FF;Amadeu de Oliveira, F;Narisawa, S;Miyake, K;Foster, BL;Millán, JL;
PMID: 34076297 | DOI: 10.1002/jbmr.4382
Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl-/- ) mice, a murine model for severe infantile HPP. Alpl-/- mice received 3 × 1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi . Treatment extended life span of Alpl-/- mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl-/- mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl-/- mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl-/- mice versus controls. Treated Alpl-/- mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP.
Brain : a journal of neurology
2022 Jul 06
Fienko, S;Landles, C;Sathasivam, K;McAteer, SJ;Milton, RE;Osborne, GF;Smith, EJ;Jones, ST;Bondulich, MK;Danby, ECE;Phillips, J;Taxy, BA;Kordasiewicz, HB;Bates, GP;
PMID: 35793238 | DOI: 10.1093/brain/awac241
Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualise HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. hom*ogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role, or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.
