Therapeutics
Abstract
Tay-Sachs and Sandhoff disease are fatal neurodegenerative diseases without an effective therapy that are caused by mutations in the HEXA and HEXB genes, respectively. Together they encode the heterodimeric isozyme of hexosaminidase (HexA) that degrades GM2 ganglioside. This report describes a 5 year-long study using a bidirectional AAV9 vector (AAV9-Bic_HexA/HexB) encoding both HEXA and HEXB in the Tay-Sachs sheep model. Bidirectional AAV9 was delivered intravenously or through various cerebral spinal fluid (CSF) delivery routes: intracerebroventricular (ICV), cisterna magna (CM) and lumbar delivery (LIT). The longest survival and best distribution were achieved by multipoint CSF delivery (combined CM, ICV and LIT) with treated animals survived up to 5 years of age (untreated Tay-Sachs animals die ~9 months). Extension in survival was accompanied by lasting improvement in neurological examination and maze testing. Improvement in biomarkers of efficacy including MRI, MR spectroscopy, diffusion tensor imaging as well as CSF levels of GM2 ganglioside and hexosaminidase A (HexA) activity was evident. Post-mortem assessments showed broad HexA distribution, GM2 ganglioside clearance and vector genome distribution, especially in deep brain structures. Therapeutic efficacy documented in this study supports translation of bidirectional vector and multipoint CSF delivery to a clinical trial in Tay-Sachs and Sandhoff disease patients.
Authors
Toloo Taghian, Jillian Gallagher, Stephanie Bertrand, William C. Baker, Kalajan Lopez Mercado, Hector R. Benatti, Erin Hall, Yvette Lopez, Abigail McElroy, John T. McCarthy, Sanjana Pulaparthi, Deborah Fernau, Samuel Mather, Sophia Esteves, Elise Diffie, Amanda Gross, Hannah G. Lahey, Xuntian Jiang, Elizabeth Parsley, Rachael Gately, Rachel Prestigiacomo, Siauna Johnson, Amanda Taylor, Lindsey Bierfeldt, Susan Tuominen, Jennifer Koehler, Guangping Gao, Jun Xie, Qin Su, Robert King, Matthew J. Gounis, Vania Anagnostakou, Ajit Puri, Ana Rita Batista, Miguel Sena-Esteves, Douglas R. Martin, Heather Gray-Edwards
Abstract
Growing evidence links human long noncoding RNAs (lncRNAs) to metabolic disease pathogenesis, yet no FDA-approved drugs target human lncRNAs. Most human lncRNAs lack conservation in other mammals, complicating efforts to define their roles and identify therapeutic targets. Here, we leveraged the concept of functionally conserved lncRNAs (FCLs) — lncRNAs that share function despite no sequence similarity — to develop a framework for identifying human lncRNAs as therapeutic targets for metabolic disorders. We used expression quantitative trait loci mapping and functional conservation analyses to pinpoint human lncRNAs influenced by disease-associated SNPs and with potential functionally conserved mouse equivalents. We identified human and mouse GULLs (glucose and lipid lowering), which regulate glucose and lipid metabolism by binding CRTC2, thereby modulating gluconeogenic genes via CREB and lipogenic genes via SREBP1. Despite their lack of sequence similarity, both lncRNAs demonstrated similar metabolic effects in obese mice, with more pronounced benefits from long-term activation. To identify druggable sites, we mapped GULLs’ binding motifs to CRTC2 (termed GULFs). Standalone human GULF, an RNA oligomer resembling FDA-approved siRNAs, significantly improved glucose and lipid levels in obese mice. This framework highlights functionally conserved human lncRNAs as promising therapeutic targets, exemplified by GULLs’ potential as a glucose- and lipid-lowering therapeutic.
Authors
Zhe Li, Sunmi Seok, Chengfei Jiang, Ping Li, Yonghe Ma, Hang Sun, Haiming Cao
Abstract
Degeneration of the neuromuscular system is a characteristic feature of spinal and bulbar muscular atrophy (SBMA), a CAG/polyglutamine (polyQ) expansion disorder caused by mutation in the androgen receptor (AR). Using a gene targeted mouse model of SBMA, AR113Q mice, we demonstrate age-dependent degeneration of the neuromuscular system that initially manifests with muscle weakness and atrophy and progresses to include denervation of neuromuscular junctions and lower motor neuron soma atrophy. Using this model, we tested the hypothesis that therapeutic intervention targeting skeletal muscle during this period of disease progression arrests degeneration of the neuromuscular system. To accomplish this, AR-targeted antisense oligonucleotides were administered subcutaneously to symptomatic AR113Q mice to reduce expression of polyQ AR in peripheral tissues but not in the spinal cord. This intervention rescued muscle atrophy, neuromuscular junction innervation, lower motor neuron soma size, and survival in aged AR113Q mice. Single-nucleus RNA sequencing revealed age-dependent transcriptional changes in the AR113Q spinal cord during disease progression which were mitigated by peripheral AR gene silencing. Our findings underscore the intricate interplay between peripheral tissues and the central nervous system in SBMA and emphasize the therapeutic effectiveness of peripheral gene knockdown in symptomatic disease.
Authors
Changwoo Lee, Zhigang Yu, Curtis J. Kuo, Leon Tejwani, Rosalie M. Grijalva, Eunwoo Bae, Hien T. Zhao, Janghoo Lim, Andrew P. Lieberman
Abstract
Genome editing has shown the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using AAV2-mediated delivery of SaCas9/sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of DFNA41, a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss (NIHL), a phenotype also observed in DFNA41 patients. Intervention at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.
Authors
Wei Wei, Wenliang Zhu, Stewart Silver, Ariel M. Armstrong, Fletcher S. Robbins, Arun Prabhu Rameshbabu, Katherina Walz, Yizhou Quan, Wan Du, Yehree Kim, Artur A. Indzhykulian, Yilai Shu, Xue-Zhong Liu, Zheng-Yi Chen
Abstract
The comorbidity of depressive symptoms in chronic pain has been recognized as a key health issue. However, whether discrete circuits underlie behavioral subsets of chronic pain and comorbid depression has not been addressed. Here, we report that dopamine 2 (D2) receptor–expressing medium spiny neurons in the nucleus accumbens medial shell (mNAcSh) mediate pain hypersensitivity and depression-like behaviors in mice after nerve injury. Two separate neural pathways mediate different symptoms. The glutamatergic inputs from the anteromedial thalamic nucleus to mNAcSh D2 neurons that innervated orexin-expressing neurons in the lateral hypothalamic area contributed to pain regulation. In contrast, the lateral septum GABAergic inputs to mNAcSh D2 neurons that disinhibit the ventral pallidum glutamatergic neurons mediated depression-like behaviors. These findings indicate the functional significance of heterogeneous mNAcSh D2 neurons and their neural pathways, providing a perspective for symptom-specific treatments of chronic pain and comorbid depression.
Authors
Di Liu, Fang-Xia Xu, Zhuang Yu, Xiao-Jing Huang, Ya-Bing Zhu, Li-Juan Wang, Chen-Wei Wu, Xu Zhang, Jun-Li Cao, Jinbao Li
Abstract
Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown clinical promise, there is still a need for therapies that overcome resistance and target non-KRASG12C mutations. KRAS activates downstream MYC, which is also a challenging-to-drug oncoprotein. We have developed an “inverted” RNAi molecule with the passenger strand of a MYC-targeting siRNA fused to the guide strand of a KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5’-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a >10-40-fold improvement in inhibiting cancer viability in vitro. When conjugated to an epidermal growth factor receptor (EGFR)-targeting ligand, the chimeric siRNA was delivered to and internalized by tumor cells. As compared with individual targeting siRNAs, the chimeric design resulted in considerably improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in multiple cancer models. This inverted chimeric design establishes proof-of-concept for ligand-directed, dual-silencing of KRAS and MYC in cancer and constitutes an innovative molecular strategy for co-targeting any two genes of interest, which has broad implications.
Authors
Yogitha S Chareddy, Hayden P. Huggins, Snehasudha S Sahoo, Lyla Stanland, Christina Gutierrez-Ford, Kristina M. Whately, Lincy Edatt, Salma H Azam, Matthew C. Fleming, Jonah Im, Alessandro Porrello, Imani Simmons, Jillian L. Perry, Albert A. Bowers, Martin Egli, Chad V. Pecot
Elevated NR2F1 underlies the persistence of invasive disease after treatment of BRAF-mutant melanoma
Abstract
Despite the clinical success of targeted inhibitors in cutaneous melanoma, therapeutic responses are transient and influenced by the aged tumor microenvironment, and drug-tolerant residual cells seed resistance. Given the similarities between drug tolerance and cellular dormancy, we studied the dormancy marker, nuclear receptor subfamily 2 group F member 1 (NR2F1), in response to targeted therapy. We utilized BRAF-V600E inhibitors (BRAFi) plus MEK inhibitors (MEKi) in BRAF-mutant melanoma models since melanoma patients treated with this combination display minimal residual disease, but ultimately tumors relapse. Transcriptomic analysis of melanoma samples from patients treated up to 20 days with BRAFi + MEKi showed increased expression of NR2F1. Similarly, NR2F1 was highly expressed in the drug-tolerant invasive cell state of minimal residual disease in patient-derived and mouse-derived xenograft tumors on BRAFi + MEKi treatment. Overexpression of NR2F1 alone was sufficient to reduce BRAFi + MEKi effects on tumor growth in vivo as well as on cell proliferation, death, and invasion in vitro. NR2F1-overexpressing cells were enriched for hallmarks gene sets for mTORC1 signaling, and NR2F1 bound to the promoter regions of genes involved in mTORC1 signaling. These cells were sensitive to the combination of BRAFi, MEKi plus rapamycin in vitro and in vivo. Melanomas from aged mice, which are known to exhibit a decreased response to BRAFi + MEKi, displayed higher levels of NR2F1 compared to tumors from young mice. Depleting NR2F1 levels in an aged mouse melanoma model improved the response to targeted therapy. These findings show high NR2F1 expression in ‘invasive-state’ residual cells and that targeting NR2F1-high cells with mTORC1 inhibitors could improve outcomes in melanoma patients.
Authors
Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L.F. Teh, Rama Kadamb, Weijia Cai, Sheera R. Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin
Abstract
Thiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.
Authors
Maud Maillard, Rina Nishii, Hieu S. Vu, Kashi R. Bhattarai, Wenjian Yang, Jing Li, Ute Hofmann, Daniel Savic, Smita Bhatia, Matthias Schwab, Min Ni, Jun J. Yang
Abstract
Nuclear size is crucial for cellular functions and often increases with malignancy. Irregular nuclei are linked to aggressive tumors, driven by genetic and epigenetic changes. However, the precise mechanisms controlling nuclear size are still not fully understood. In this study, we demonstrated that cancer-associated speckle-type POZ protein (SPOP) mutations enlarged nuclear size by reducing the protein level of lamin B2 (LMNB2), a key nuclear integrity protein. Mechanistically, SPOP bound to LMNB2 and promoted its mono-ubiquitination at lysine-484, which protected it from degradation by the E3 ubiquitin ligase WD repeat domain 26. SPOP mutations disrupted this process, leading to reduced LMNB2 levels and impaired nuclear envelope (NE) integrity. This compromised NE was more vulnerable to damage from farnesyltransferase inhibitors (FTIs), causing nuclear rupture in SPOP-mutant tumor cells. This study identified SPOP as a positive regulator of nuclear size; the findings suggest tumors with SPOP mutations may be vulnerable to FTI-based therapies.
Authors
Zixi Wang, Lei Li, Qi Ye, Yuzeshi Lei, Mingming Lu, Leihong Ye, Jialu Kang, Wenyue Huang, Shan Xu, Ke Wang, Jing Liu, Yang Gao, Chenji Wang, Jian Ma, Lei Li
Abstract
Contemporary cancer treatment strategies are shifting toward targeted therapies to improve efficacy and minimize toxicity. Here, we report the design and preclinical evaluation of MBRC-101, a first-in-class antibody-drug conjugate (ADC) targeting EphA5, a receptor tyrosine kinase with an established role in embryonic development but not extensively studied in cancer. We show that EphA5 is expressed in multiple solid tumors, including cancers of the aerodigestive (non–small cell lung, head and neck, gastric, colon, and pancreatic) and genitourinary (bladder and ovary) tracts, as well as most breast cancer subsets (including triple-negative tumors), with limited expression in normal tissues. MBRC-101 is a humanized anti-EphA5 antibody conjugated to monomethyl auristatin E (MMAE) through a ThioBridge, thereby ensuring stable drug-to-antibody ratio and reducing off-target effects. MBRC-101 showed potent antitumor activity, achieving complete tumor regression in several patient-derived xenograft models. Preclinical Good Laboratory Practice–compliant toxicology studies in rats and nonhuman primates demonstrated that MBRC-101 is well tolerated, with observed toxicities limited to known MMAE off-target effects. These findings establish EphA5 as a therapeutic target in cancer and support the translational development of MBRC-101 as a promising ADC candidate for clinical evaluation, currently in a first-in-human multicenter investigational trial for patients with advanced solid tumors (ClinicalTrials.gov, NCT06014658).
Authors
Fernanda I. Staquicini, Fenny H.F. Tang, Vanessa de Oliveira, Sun-Young Kim, Ethan R. Chen, Christopher Markosian, Daniela I. Staquicini, Yongjian Wu, J. Kellogg Parsons, Kirstin F. Barnhart, Stephen C. Alley, Isan Chen, Wadih Arap, Renata Pasqualini
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ISSN: 0021-9738 (print), 1558-8238 (online)