Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Dr. Jixue Li, Dr. Yanjie Li, Dr. Jun Wang, Dr. Trevor J. Gonzalez, Dr. Aravind Asokan, Dr. Jill Napierala, and Dr. Marek Napierala; Hum Gene Ther. 2020;10.1089/hum.2020.053. doi:10.1089/hum.2020.053

Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site-specifically integrated into the genome of HEK293T and human induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

Treatment of Dilated Cardiomyopathy in a Mouse Model of Friedreich’s Ataxia using N-acetylcysteine and Identification of Alterations in MicroRNA Expression that Could be Involved in its Pathogenesis

S. Chiang, M.L.H. Huang, D.R. Richardson, Pharmacological Research, 2020, 104994, doi:10.1016/j.phrs.2020.104994.

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

Vestibular Impact of Friedreich Ataxia in Early Onset Patients

A Maudoux, N Teissier, M Francois, Th Van Den Abbeele, C Alberti, I Husson, S R Wiener-Vacher; Cerebellum Ataxias. 2020;7:6. Published 2020 May 28. doi:10.1186/s40673-020-00115-z

We show that Friedreich ataxia, even at onset, frequently associate saccadic intrusions, abnormal ABRs and decreased vestibulo-ocular and vestibulospinal responses progressing over time. These sensory impairments combined with ataxia further impair patient's autonomy. These vestibular, auditory and visual impairments could be used as markers of the severity and progression of the disease. Adding vestibular and auditory testing to Friedreich patient's evaluation may help physicians improve patient's management.

A Novel Solution-Gated Graphene Transistor Biosensor for Ultrasensitive Detection of Trinucleotide Repeats

Zhiqi Ge, Mingyu Ma, Zang Chang, Meijun Chen, Hanping He, Xiuhua Zhang and Shengfu Wang, 2020, doi:10.1039/D0AN00205D

A new way to detect GAA trinucleotide repeats (TNR) based on solution-gated graphene transistor (SGGT) with high performance was developed. Friedreich's ataxia (FRDA) is a neurodegenerative disease where the first intron of the frataxin (FXN) gene exhibits an extended GAA repeat region. Herein, a SGGT biosensor was constructed based on G-quadruplex DNAzymes and graphene channels. The DNAzymes quantify the captured target DNA by producing a strong catalytic current signal depending on the peroxidase-like activity. The higher the target DNA quantity captured on the gate electrode is, the higher is the concentration of DNAzymes on the surface of the gate electrode, which generates a high catalytic current. Due to the excellent self-amplifying performance of the transistor, the current signal of the SGGT is several hundreds of times larger than in conventional electrochemistry under identical detection conditions. Moreover, a large current signal can be obtained in the case of a low concentration of H2O2 when compared to the case of an enzyme-catalyzed transistor. The SGGT biosensor also exhibits ultra-low detection limit (32.25 fM), a wide linear range (100 fM–100 nM), and excellent selectivity. The results show that the SGGT biosensor shows a great potential in the early diagnosis of neurodegenerative diseases.

Cerebellar cognitive disorder parallels cerebellar motor symptoms in Friedreich ataxia

Naeije, G., Rai, M., Allaerts, N., Sjogard, M., De Tiège, X. and Pandolfo, M.; Ann Clin Transl Neurol. doi:10.1002/acn3.51079

Dentate nuclei (DN) are involved in cerebellar modulation of motor and cognitive functions, whose impairment causes ataxia and cerebellar cognitive affective syndrome (CCAS). Friedreich ataxia (FRDA) disease progression relates to degeneration of the dentate nucleus and dentato‐thalamic pathways, causing cerebellar ataxia. Volumetric MRI also shows mild loss in the cerebellar cortex, brainstem, and motor cortex. Cognitive deficits occur in FRDA, but their relationship with ataxia progression is not fully characterized. We found a significant positive correlation between severity of patients’ ataxia and more marked CCAS as assessed with the CCAS‐Scale. This relation could be related to progressive DN impairment.

HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich’s Ataxia

Sherzai, Mursal; Valle, Adamo; Perry, Nicholas; Kalef-Ezra, Ester; Al-Mahdawi, Sahar; Pook, Mark; Sara Anjomani Virmouni. Frontiers. Collection. doi:10.3389/fgene.2020.00584

Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

Functional coupling of presequence processing and degradation in human mitochondria

Kücükköse, C., Taskin, A.A., Marada, A., Brummer, T., Dennerlein, S. and Vögtle, F.‐N. (2020), FEBS J. doi:10.1111/febs.15358

The mitochondrial proteome is built and maintained mainly by import of nuclear‐encoded precursor proteins. Most of these precursors use N‐terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce.

Significance of NT-proBNP and High-sensitivity Troponin in Friedreich Ataxia

Legrand, L.; Maupain, C.; Monin, M.-L.; Ewenczyk, C.; Isnard, R.; Alkouri, R.; Durr, A.; Pousset, F.; J. Clin. Med. 2020, 9, 1630. doi:10.3390/jcm9061630

Conclusion: hsTnT was increased in 1/3 of the adult FA and associated with increased septal wall thickness. Increased NT-proBNP remained a marker of increased left ventricular filling pressure. This could be used to identify patients that should undergo a closer cardiac surveillance.

Efficacy and Safety of CSF- Delivered AVXS-401 in Mice and NonHuman Primates for the Treatment of Friedreich’s Ataxia

Martin Fugere, Rajeev Sivasankaran, Susan McQuown, Chang Choi, Katrina Salvador, Shirley Phillips, Yin Gu, Binh Chu, Janet Do, Tsun-Kai Chang, Katherine Nguyen, Yanchi Li, Stephanie Solano, Natalia Teider, Colin Fennelly, Ricardo Dolmetsch, Monica Bennett, Kevin Foust, Page Bouchard; AveXis Research and Development, San Diego, CA, Novartis Institutes for Biomedical Research, Cambridge, MA

For clinical translation, we developed AVXS-401, a self-complementary adenoassociated virus (AAV9) based gene replacement therapy to provide sustained expression of FXN in key tissues relevant to FA. Toxicity studies in wildtype mice proved AVXS-401 is safe and well-tolerated. Dose ranging efficacy studies following a one-time, pre-symptomatic intracerebroventricular (ICV) administration of AVXS-401 in conditional FXN-deficient mice in the CNS (Pvalb) demonstrates amelioration of behavioral phenotypes, rescue of Purkinje neurons and cerebellar gliosis at low doses. ICV delivery of AVXS-401 in cardiac mutants (MCK) results in full recovery of cardiac functions as measured by magnetic resonance imaging (MRI), prevention of histopathological evidence of cardiomyopathy and >300% increase in median survival at efficacious doses.
Scale up of therapeutic doses to non-human primates (NHP) showed that AVXS-401 is safe and well tolerated with no aberrant behavior, clinical or anatomical pathology attributable to frataxin expression. Importantly, AVXS-401 provides durable mRNA transcription in the CNS and heart of NHP at 6 months postinjection with frataxin expression detectable above endogenous levels. Dose escalation studies by intrathecal (IT) administration in NHP show a dose correlation between mice and NHP by ddPCR quantification of vector genomes. Together these pre-clinical data show that AVXS-401 is suitable for first-inhuman studies.

PTC announced delays in its gene therapy program for Friedreich’s ataxia

MAY 19, 2020. PTC announced delays in its gene therapy program for Friedreich’s ataxia and in the opening of a manufacturing facility in New Jersey, which is now expected to begin producing clinical material early next year.