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Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

ABSTRACT: Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA (pri-miRNA) scaffolds impact the potency and safety profile of the expressed amiR in vivo. In our process to develop an AAV amiR targeting SOD1, we performed a preclinical characterization of two pri-miRNA scaffolds, miR155 and miR30a, sharing the same guide strand sequence. We report that, while the miR155-based vector, compared with the miR30a-based vector, leads to a higher level of the amiR and more robust suppression of SOD1 in vitro and in vivo, it also presents significantly greater risks for CNS-related toxicities in vivo. Despite miR30a-based vector showing relatively lower potency, it can significantly delay the development of ALS-like phenotypes in SOD1-G93A mice and increase survival in a dose-dependent manner. These data highlight the importance of scaffold selection in the pursuit of highly efficacious and safe amiRs for RNA interference gene therapy.

SUBMITTER: Chen SK

PROVIDER: S-EPMC10622307 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

Chen Shukkwan K SK Hawley Zachary C E ZCE Zavodszky Maria I MI Hana Sam S Ferretti Daniel D Grubor Branka B Hawes Michael M Xu Shanqin S Hamann Stefan S Marsh Galina G Cullen Patrick P Challa Ravi R Carlile Thomas M TM Zhang Hang H Lee Wan-Hung WH Peralta Andrea A Clarner Pete P Wei Cong C Koszka Kathryn K Gao Feng F Lo Shih-Ching SC

Molecular therapy. Nucleic acids 20231016

Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA ...[more]

PMID: 37928442

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Project description:IntroductionAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that widely affects motor neurons of the CNS. About 20% of patients with ALS have familial ALS (fALS). One of the classic models of ALS are SOD1G93A mice. Misfolded SOD1 protein can be overexpressed in motor neurons, which results in progressive paralysis of the limbs of mice. There is still no effective treatment for ALS. In recent years, the treatment of ALS by regulating autophagy has become a research hotspot. Autophagy obstacles have been confirmed to be one of the early pathological events of ALS. Rab7 is a member of the Ras superfamily and plays a key role in the late stage of autophagy. In our previous studies, we found that prenoylation of Rab7 was inhibited in the ALS model. Prenylation is a post-translational modification in which farnesyl or geranylgeranyl groups are covalently linked to target proteins. Based on these findings, we proposed the novel idea that the regulation of RabGGTB (the β-subunit of RabGGTase) mediated prenylation modification of Rab7, and that this can be used as a prevention and treatment of ALS associated with abnormal protein accumulation.MethodsIn the present study, RabGGTB was overexpressed in mouse spinal cord motoneurons by using adeno-associated virus as vector. Then immunofluorescence quantitative analysis was used for pathological study. The body weight, footprint analysis, the accelerating rotarod test, and neurological deficits score were used to evaluate animal behavior.ResultsOur results show that the protein level of RabGGTB was significantly increased in the lumbar and thoracic regions of spinal cord motoneurons of injected mice. Furthermore, the onset time and survival time of SOD1G93A mice injected with AAV9-RabGGTB-GFP+ were delayed compared with those of mice without overexpression. At the same time, we also observed a decrease in SOD1 misfolded and glial overactivation in the lumbar spinal cord of these SOD1G93A mice.ConclusionThe findings reported here show that RabGGTB plays a significant role in the pathogenesis of SOD1G93A mice and with great therapeutic potential for reducing abnormal aggregation of SOD1 in ALS.

Project description:The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism.

Project description:BackgroundMutations in SOD1 cause 13% of familial amyotrophic lateral sclerosis. In the SOD1 Gly93Ala rat model of amyotrophic lateral sclerosis, the antisense oligonucleotide ISIS 333611 delivered to CSF decreased SOD1 mRNA and protein concentrations in spinal cord tissue and prolonged survival. We aimed to assess the safety, tolerability, and pharmacokinetics of ISIS 333611 after intrathecal administration in patients with SOD1-related familial amyotrophic lateral sclerosis.MethodsIn this randomised, placebo-controlled, phase 1 trial, we delivered ISIS 333611 by intrathecal infusion using an external pump over 11·5 h at increasing doses (0·15 mg, 0·50 mg, 1·50 mg, 3·00 mg) to four cohorts of eight patients with SOD1-positive amyotrophic lateral sclerosis (six patients assigned to ISIS 333611, two to placebo in each cohort). We did the randomisation with a web-based system, assigning patients in blocks of four. Patients and investigators were masked to treatment assignment. Participants were allowed to re-enrol in subsequent cohorts. Our primary objective was to assess the safety and tolerability of ISIS 333611. Assessments were done during infusion and over 28 days after infusion. This study was registered with Clinicaltrials.gov, number NCT01041222.FindingsSeven of eight (88%) patients in the placebo group versus 20 of 24 (83%) in the ISIS 333611 group had adverse events. The most common events were post-lumbar puncture syndrome (3/8 [38%] vs 8/24 [33%]), back pain (4/8 [50%] vs 4/24 [17%]), and nausea (0/8 [0%] vs 3/24 [13%]). We recorded no dose-limiting toxic effects or any safety or tolerability concerns related to ISIS 333611. No serious adverse events occurred in patients given ISIS 333611. Re-enrolment and re-treatment were also well tolerated.InterpretationThis trial is the first clinical study of intrathecal delivery of an antisense oligonucleotide. ISIS 333611 was well tolerated when administered as an intrathecal infusion. Antisense oligonucleotides delivered to the CNS might be a feasible treatment for neurological disorders.FundingThe ALS Association, Muscular Dystrophy Association, Isis Pharmaceuticals.

Dataset Information

Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

Publications

Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

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