Preclinical studies of gene replacement therapy for CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth and dendritic morphogenesis as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (i.c.v.) injection resulted in broader, more optimal biodistribution than did intra-cisterna magna (i.c.m.) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provide proof of concept that i.c.v. delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses, suggesting a requirement for broad distribution for efficacy.

Phase Ib Study of Unesbulin (PTC596) Plus Dacarbazine for the Treatment of Locally Recurrent, Unresectable or Metastatic, Relapsed or Refractory Leiomyosarcoma.

PURPOSE: This multicenter, single-arm, open-label, phase Ib study was designed to determine the recommended phase II dose (RP2D) and to evaluate the safety and preliminary efficacy of unesbulin plus dacarbazine (DTIC) in patients with advanced leiomyosarcoma (LMS). PATIENTS AND METHODS: Adult subjects with locally advanced, unresectable or metastatic, relapsed or refractory LMS were treated with escalating doses of unesbulin orally twice per week in combination with DTIC 1,000 mg/m2 intravenously (IV) once every 21 days. The time-to-event continual reassessment method was used to determine the RP2D on the basis of dose-limiting toxicities (DLTs) assessed during the first two 21-day treatment cycles. All explored doses of unesbulin (200 mg up to 400 mg) were in combination with DTIC. An expansion cohort was enrolled to evaluate the safety and efficacy of unesbulin at the RP2D. RESULTS: Unesbulin 300 mg administered orally twice per week in combination with DTIC 1,000 mg/m2 IV once every 21 days was identified as the RP2D. On the basis of data from 27 subjects who were deemed DLT-evaluable, toxicity was higher in the unesbulin 400 mg group, with three of four subjects (75%) experiencing DLTs versus one of four subjects (25%) in the 200 mg group and three of 19 subjects (15.8%) in the 300 mg group. The most commonly reported DLTs and treatment-related grade 3 and 4 adverse events were thrombocytopenia and neutropenia. At the RP2D, seven subjects who were efficacy evaluable achieved partial response for an objective response rate of 24.1%. CONCLUSION: Unesbulin 300 mg twice per week plus DTIC 1,000 mg/m2 once every 21 days was identified as the RP2D, demonstrating a favorable benefit-risk profile in a heavily pretreated population of adults with advanced LMS.

Tumor Microenvironment Landscapes Supporting EGFR-mutant NSCLC Are Modulated at the Single-cell Interaction Level by Unesbulin Treatment.

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.

SAR studies toward discovery of emvododstat (PTC299), a potent dihydroorotate dehydrogenase (DHODH) inhibitor.

Dihydroorotate dehydrogenase (DHODH) is the enzyme that catalyzes a rate-determining step during the de novo synthesis of uridine, an important source of cellular pyrimidine nucleotides. Ability to modulate the activity of this enzyme may be used to control diseases associated with rapid, out-of-control cell growth in oncology, immunology, and virology. Emvododstat (PTC299) is a tetrahydro-ß-carboline DHODH inhibitor discovered through the GEMS technology (Gene Expression Modulation by Small-Molecules). Described in this paper is the lead optimization campaign that culminated in the discovery of this highly potent DHODH inhibitor.

Emvododstat, a Potent Dihydroorotate Dehydrogenase Inhibitor, Is Effective in Preclinical Models of Acute Myeloid Leukemia.

Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias.

In vitro metabolism, pharmacokinetics and drug interaction potentials of emvododstat, a DHODH inhibitor.

Emvododstat was identified as a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. The objective of this paper is to evaluate the metabolism, pharmacokinetics, and drug interaction potentials of emvododstat.Emvododstat showed high binding to plasma protein with minimal distribution into blood cells in mouse, rat, dog, monkey, and human whole blood.O-Demethylation followed by glucuronidation appeared to be the major metabolic pathway in rat, dog, monkey, and human hepatocytes. CYP2C8, 2C19, 2D6, and 3A4 were involved in O-desmethyl emvododstat metabolite formation. Both emvododstat and O-desmethyl emvododstat inhibited CYP2D6 activity and induced CYP expression to different extents in vitro.Emvododstat and O-desmethyl emvododstat inhibited BCRP transporter activity but did not inhibit bile salt transporters and other efflux or uptake transporters. Neither emvododstat nor O-desmethyl emvododstat was a substrate for common efflux or uptake transporters investigated.Emvododstat is bioavailable in mice, rats, dogs, and monkeys following a single oral dose. The absorption was generally slow with the mean plasma Tmax ranging from 2 to 5 h; plasma exposure of O-desmethyl emvododstat was lower in rodents, but relatively higher in dogs and monkeys.

Preclinical and Early Clinical Development of PTC596, a Novel Small-Molecule Tubulin-Binding Agent.

PTC596 is an investigational small-molecule tubulin-binding agent. Unlike other tubulin-binding agents, PTC596 is orally bioavailable and is not a P-glycoprotein substrate. So as to characterize PTC596 to position the molecule for optimal clinical development, the interactions of PTC596 with tubulin using crystallography, its spectrum of preclinical in vitro anticancer activity, and its pharmacokinetic-pharmacodynamic relationship were investigated for efficacy in multiple preclinical mouse models of leiomyosarcomas and glioblastoma. Using X-ray crystallography, it was determined that PTC596 binds to the colchicine site of tubulin with unique key interactions. PTC596 exhibited broad-spectrum anticancer activity. PTC596 showed efficacy as monotherapy and additive or synergistic efficacy in combinations in mouse models of leiomyosarcomas and glioblastoma. PTC596 demonstrated efficacy in an orthotopic model of glioblastoma under conditions where temozolomide was inactive. In a first-in-human phase I clinical trial in patients with cancer, PTC596 monotherapy drug exposures were compared with those predicted to be efficacious based on mouse models. PTC596 is currently being tested in combination with dacarbazine in a clinical trial in adults with leiomyosarcoma and in combination with radiation in a clinical trial in children with diffuse intrinsic pontine glioma.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

Pharmacokinetics and Safety of PTC596, a Novel Tubulin-Binding Agent, in Subjects With Advanced Solid Tumors.

PTC596 is a novel, orally bioavailable, small-molecule tubulin-binding agent that reduces B-cell-specific Moloney murine leukemia virus insertion site 1 activity and is being developed for the treatment of solid tumors. A phase 1, open-label, multiple-ascending-dose study was conducted to evaluate the pharmacokinetics and safety of the drug in subjects with advanced solid tumors. PTC596 was administered orally biweekly based on body weight. Dose escalation followed a modified 3 + 3 scheme using doses of 0.65, 1.3, 2.6, 5.2, 7.0, and 10.4 mg/kg. Following oral administration, PTC596 was rapidly absorbed, and between 0.65 and 7.0 mg/kg reached a maximum plasma concentration 2 to 4 hours after dosing. Area under the plasma concentration-time curve increased proportionally with body weight-adjusted doses. Maximum plasma concentration increased with dose, although the increase was less than dose proportional at dose levels >2.6 mg/kg. No accumulation occurred after multiple administrations up to 7.0 mg/kg. PTC596 had a terminal half-life ranging 12 to 15 hours at all doses except for the highest dose of 10.4 mg/kg, where the half-life was approximately 20 hours. Overall, PTC596 was well tolerated. The most frequently reported PTC596-related treatment-emergent adverse events were mild to moderate gastrointestinal symptoms, including diarrhea (54.8%), nausea (45.2%), vomiting (35.5%), and fatigue (35.5%). Only 1 patient treated with 10.4 mg/kg experienced dose-limiting toxicity of neutropenia and thrombocytopenia, both of which were reversible. Stable disease as best overall response was observed among 7 patients, with 2 patients receiving the study drug up to 16 weeks. These results support the further development of PTC596 for the treatment of solid tumors.

The combination of the tubulin binding small molecule PTC596 and proteasome inhibitors suppresses the growth of myeloma cells.

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.

The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene.

Future progress in the treatment of multiple myeloma (MM) requires both the characterisation of key drivers of the disease and novel, innovative approaches to tackle these vulnerabilities. The present study focussed on the pre-clinical evaluation of a novel drug class, BMI-1 modulators, in MM. We demonstrate potent activity of PTC-028 and PTC596 in a comprehensive set of in vitro and in vivo models, including models of drug resistance and stromal support. Treatment of MM cells with PTC-028 and PTC596 downregulated BMI-1 protein levels, which was found to correlate with drug activity. Surprisingly, BMI-1 was dispensable for the activity of BMI-1 modulators and MM cell growth. Our data rather point to mitotic arrest accompanied by myeloid cell leukaemia-1 (MCL-1) loss as key anti-MM mechanisms and reveal impaired MYC and AKT signalling activity due to BMI-1 modulator treatment. Moreover, we observed a complete eradication of MM after PTC596 treatment in the 5TGM.1 in vivo model and define epigenetic compounds and B cell leukaemia/lymphoma 2 homology domain 3 (BH3) mimetics as promising combination partners. These results bring into question the postulated role of BMI-1 as an essential MM gene and confirm BMI-1 modulators as potent anti-mitotic agents with encouraging pre-clinical activity that supports their rapid translation into clinical trials.

Effective Delivery of a Microtubule Polymerization Inhibitor Synergizes with Standard Regimens in Models of Pancreatic Ductal Adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that is broadly chemoresistant, due in part to biophysical properties of tumor stroma, which serves as a barrier to drug delivery for most classical chemotherapeutic drugs. The goal of this work is to evaluate the preclinical efficacy and mechanisms of PTC596, a novel agent with potent anticancer properties in vitro and desirable pharmacologic properties in vivo.Experimental Design: We assessed the pharmacology, mechanism, and preclinical efficacy of PTC596 in combination with standards of care, using multiple preclinical models of PDA. RESULTS: We found that PTC596 has pharmacologic properties that overcome the barrier to drug delivery in PDA, including a long circulating half-life, lack of P-glycoprotein substrate activity, and high systemic tolerability. We also found that PTC596 combined synergistically with standard clinical regimens to improve efficacy in multiple model systems, including the chemoresistant genetically engineered "KPC" model of PDA. Through mechanistic studies, we learned that PTC596 functions as a direct microtubule polymerization inhibitor, yet a prior clinical trial found that it lacks peripheral neurotoxicity, in contrast to other such agents. Strikingly, we found that PTC596 synergized with the standard clinical backbone regimen gemcitabine/nab-paclitaxel, yielding potent, durable regressions in a PDX model. Moreover, similar efficacy was achieved in combination with nab-paclitaxel alone, highlighting a specific synergistic interaction between two different microtubule-targeted agents in the setting of pancreatic ductal adenocarcinoma. CONCLUSIONS: These data demonstrate clear rationale for the development of PTC596 in combination with standard-of-care chemotherapy for PDA.

Acute myeloid leukemia driven by the CALM-AF10 fusion gene is dependent on BMI1.

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are often ineffective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10-positive leukemias which may lay the foundation for novel targeted therapies. Here we demonstrate that the Polycomb Repressive Complex 1 gene BMI1 is consistently overexpressed in adult and pediatric CALM-AF10-positive leukemias. We demonstrate that genetic Bmi1 depletion abrogates CALM-AF10-mediated transformation of murine hematopoietic stem and progenitor cells (HSPCs). Furthermore, CALM-AF10-positive murine and human AML cells are sensitive to the small-molecule BMI1 inhibitor PTC-209 as well as to PTC-596, a compound in clinical development that has been shown to result in downstream degradation of BMI1 protein. PTC-596 significantly prolongs survival of mice injected with a human CALM-AF10 cell line in a xenograft assay. In summary, these results validate BMI1 as a bona fide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements.

Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties.

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.

BMI1 is a therapeutic target in recurrent medulloblastoma.

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.

The minor gentamicin complex component, X2, is a potent premature stop codon readthrough molecule with therapeutic potential.

Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92-99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called "designer" aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent.