Events

Shahragim Tajbakhsh: Curriculum Vitae

Abstract
The advent of genome editing technologies, including the RNA-guided CRISPR/Cas9 system, has enabled the precise editing of endogenous human genes. For example, we engineered CRISPR/Cas9-based nucleases to correct the mutated human dystrophin gene in cells isolated from Duchenne muscular dystrophy (DMD) patients. When delivered directly to a mouse model of this disease, gene editing by the CRISPR/Cas9 system led to gene restoration and improvement of biochemical and mechanical muscle function. Genome editing and dystrophin protein restoration is sustained in the mdx mouse model of DMD for one year after a single intravenous administration of AAV-CRISPR. We also confirmed immunogenic host response to Cas9 when administered via AAV vectors to adult mice and observed unintended genome and transcript alterations induced by AAV-CRISPR. We have observed gene editing in muscle satellite cells following AAV-CRISPR delivery in vivo, supporting the possibility of long-term gene correction despite muscle cell turnover. More recently, we have developed novel humanized models of this disease for the preclinical development of therapies that will correct human disease-causing mutations. New constructs have been developed and validated for significant levels of gene correction and dystrophin restoration in this model. Moreover, we have demonstrated in vivo gene editing that restores a full-length dystrophin gene, in contrast to previous approaches that restore a truncated, partially functional protein. These studies demonstrate the potential for genome editing to be used to treat DMD and other neuromuscular disorders, and also highlight issues for further study and development.

Biography:
Dr. Charles A. Gersbach is the John W. Strohbehn Distinguished Professor of Biomedical Engineering at Duke University, the Director of the Duke Center for Biomolecular and Tissue Engineering, and the Director of the Duke Center for Advanced Genomic Technologies. His research interests are in genome and epigenome editing, gene therapy, regenerative medicine, biomolecular and cellular engineering, synthetic biology, and genomics. His work has led to new approaches to study genome structure and function, program cell biology, and treat genetic disease. Dr. Gersbach’s work has been recognized through awards including the NIH Director’s New Innovator Award, the NSF CAREER Award, the Outstanding New Investigator Award from the American Society of Gene and Cell Therapy, and induction as a Fellow of the American Institute for Medical and Biological Engineering. He is also the co-founder of three biotechnology companies and an advisor to several others.

In my talk, I will focus on two distal myopathies which are apparently relatively frequent in Japan: GNE myopathy and oculopharyngodistal myopathy (OPDM).

GNE myopathy is an autosomal recessive muscle disease characterized clinically by preferential involvement of tibialis anterior muscle and relative sparing of quadriceps, and pathologically by the presence of rimmed vacuoles, which is caused mostly by missense mutations in the GNE gene that encodes a protein with the activity of two enzymes in sialic acid biosynthesis, UDP-GlcNAC 2-epimerase and ManNAc kinase, resulting in the reduction of the sialic acid levels in serum and skeletal muscles. So far, 103 different mutations have been identified among 345 Japanese unrelated families. Interestingly, we have identified only 3 patients homozygous for the second most common mutation p.D207V as well as a healthy homozygote. Furthermore, the allele frequency of p.D207V is higher than that of the most common mutation in general Japanese population, raising a possibility that p.D207V may well be a mild mutation whose homozygosity does not lead to disease phenotype in most cases. In fact, based upon the results of biochemical assay, intra- and inter molecular ManNAc transfer in GNE molecules with homozygous p.D207V mutations are mildly decreased, leading to borderline sialic acid production. We treated our model mice, which showed a phenotype clinicopathologically similar to human patiens, with ManNAc, NeuAc, and sialic acid conjugate, sialyllactose from around 15 weeks of age and continued to around 55 weeks. Phenotypic manifestations were almost completely suppressed, indicating that sialic acid deficiency is the cause of GNE myopathy and that the disease can be suppressed by sialic acid supplementation.

OPDM is an autosomal dominant muscle disease which is characterized by ocular and bulbar symptoms, including ptosis, ophtalmoparesis, and dysphagia, in addition to preferential distal limb muscle involvement. It is clinicopathologically similar to ocuolopharyngeal muscular dystrophy (OPMD) which is caused by alanine codon expansions in PABPN1. Recently, our collaborator identified the expansion of the CGG repeats in the 5’ UTR of LRP12 in OPDM patients as well as of NOTCH2NCL in patients with neuronal intranuclear inclusion disease (NIID). Soon after that, similar 5’ UTR CGG repeat expansions in GIPC1 and NOTCH2NLC were also associated with OPDM. In Japan, majority of OPDM patients seem to have expansions in LRP12 while Chinese OPDM patients those in GIPC1. I will discuss detailed clinicopathological features of OPDM_LRP12 and pathological differentiation between OPDM and OPMD.

Biography

Dr. Ichizo Nishino is Director of Department of Neuromuscular Research, National Institute of Neuroscience (NIN), National Center of Neurology and Psychiatry (NCNP). He is also appointed to Director of two departments in Medical Genome Center in NCNP. Currently, he is a Guest/Visiting Professor at 4 universities: University of Yamanashi, Kaohsiung Medical University (Taiwan), Peking University (China) and Siriraj Hospital, Mahidol University (Thailand).

Dr. Nishino obtained his M.D. in 1989 from Kyoto University. After 5 years of neurology training supervised by Prof. Jun Kimura, he started muscle disease research in NCNP in 1994 under the supervision of Dr. Ikuya Nonaka, and further continued research for 2 years in Columbia University where his research was guided by Profs. Michio Hirano and Salvatore DiMauro. He was appointed to his current position in 2001. By now, he has published more than 600 PubMed-listed papers in the field.

His laboratory functions as a referral center for muscle disease in Japan, providing diagnostic service for muscle pathology and genetic analysis. His lab is designated as national NGS center for hereditary muscle disease by AMED (governmental funding organization in Japan). His lab collects around 80% of muscle biopsies performed in Japan (1103 cases in 2021) and he signs out all cases. As a result, his muscle repository has more than 22,000 frozen muscle biopsy samples, which is one of the largest collections of the patient’s muscles.

More information on Florent Ginhoux

Walter R. Frontera, MD, PhD, is Professor in the Departments of Physical Medicine, Rehabilitation, and Sports Medicine (PM&R) and Physiology and Biophysics at the University of Puerto Rico (UPR).  Dr. Frontera completed his MD in 1979 and a residency in PM&R in 1983 at the UPR and a doctoral degree in applied anatomy and physiology at Boston University in 1986. During the last 35 years he has served as Professor and Inaugural Chair of PM&R at the UPR, Harvard Medical School and Vanderbilt University School of Medicine as well as Dean of the Faculty of Medicine at the UPR.  In 1995 he was Visiting Professor at the at the Karolinska Hospital in Stockholm, Sweden in the Department of Clinical Neurophysiology.  He received a Master of Arts degree (Hon. Causa) from Harvard University in 2004.

His main research interest is geriatric rehabilitation and in particular the study of the mechanisms underlying muscle atrophy and weakness in elderly.  Based on his studies of human sarcopenia, Dr. Frontera has developed rehabilitative interventions using therapeutic exercise to slow down and/or reverse skeletal muscle alterations associated with advanced adult age.  In 2005, he was the Chair of the Program Committee for the US National Research Summit on Building Capacity in PM&R. He has more than 275 i-scientific publications including 111 peer-reviewed articles and 12 edited/co-edited books (including the leading textbook in the specialty of PM&R). His books have been translated into Chinese, Italian, Korean, Portuguese, and Spanish.

Currently, Dr. Frontera serves as the Editor-in-Chief of The American Journal of PM&R. In 2008 he was elected member of the National Academy of Medicine of the US National Academies. He is an elected fellow of the American Association for the Advancement of Science, the Royal College of Physicians (London), and the American Physiological Society). He was appointed honorary professor at the Nanjing Medical University in China in 2018.  Dr. Frontera has presented more than 400 invited lectures in 59 countries and served as a grant reviewer and graduate research examiner for Universities in Canada, South Africa and Hong Kong.

Dr. Frontera has been very active at the US National Institutes of Health as a permanent member of the Geriatrics and Rehabilitation Medicine, Musculoskeletal Rehabilitation Sciences, and Function and Rehabilitation Sciences scientific review panels. He also served on the Blue-Ribbon Panel appointed by the Director of the NIH to assess the status of rehabilitation research across NIH. Dr. Frontera has received several awards including the Association of Academic Physiatrist’s Distinguished Academician Award, the Best Scientific Research Paper (3 times) presented by the American Academy of PM&R, the Excellence in Rehabilitation of Aging Persons Award of the Gerontological Society of America and the Distinguished Member Award of the American Academy of PM&R. He is an honorary member of national societies of PM&R in Belgium, Chile, Dominican Republic, Italy, Mexico, and Japan.  

A complete list of published work can be found at –  https://www.ncbi.nlm.nih.gov/pubmed/?term=frontera%2C+walter

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Antisense oligomer induced spliceoform switching:  from Duchenne muscular dystrophy to common serious inherited and acquired diseases

Steve Wilton ^1,2*, Merlin Thomas³, May Aung-Htut^1,2, Craig McIntosh^1,2, Abbie Adams^1,2, Kristin Ham^1,2, Russell Johnsen², Dunhui Li^1,2, Jessica Cale², Bal Poudel^1,2

¹Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Australia. ²Perron Institute, The University of Western Australia, Australia. ³Department of Diabetes, Monash University, Australia.

* email:

Abstract

Duchenne muscular dystrophy is an X-linked recessive disease with a high de novo mutation rate. Arising from null mutations (nonsense, frame-shifting insertions/deletions varying from a single base to multiple exons) that preclude synthesis of a functional dystrophin protein, affected boys are typically restricted to a wheelchair before the age of 12 years.  In contrast, in-frame whole exon deletions in the massive 79 exon, 14 kb mRNA dystrophin gene can present with no or only mild symptoms, indicating that many exons in this gene are not essential for near normal function.  This phenotype:genotype correlation laid the foundations for an antisense oligomer intervention during the dystrophin pre-mRNA processing. This intervention aimed to excise a specific exon during splicing to restore the reading frame around a frame-shifting deletion, or remove an intra-exonic nonsense mutation without disrupting the reading frame. The US Food and Drug Administration have now granted accelerated approval for three morpholino oligomers targeting different dystrophin exons, each relevant to certain sub-classes of genomic deletions.  The first drug approved in September 2016, (Eteplirsen), induces skipping of exon 51 and is relevant to the 10-13% of DMD patients whose genomic deletions ended with exon 50 or with DMD-causing deletions starting with exon 52.  Casimersen, the last drug approved in February 2021 targets exon 45 and is relevant to various deletions ending with exon 44 or starting with exon 46. Trials are ongoing evaluating higher doses, treating younger children and examining the “second generation morpholinos”, and eteplirsen coupled to a cell penetrating peptide that is administered monthly at lower doses. Results reported to date are most promising, with monthly injections of 30mg/kg (rather than weekly) resulting in 8-fold more dystrophin than the uncomplexed Eteplirsen !

Splice switching antisense oligomers are now offering some hope in the treatment of rare diseases and much has been learnt.  The Molecular Therapy laboratory in Perth is exploring possible antisense oligomer strategies for many different diseases, from juvenile onset Parkinson’s to adult onset Pompe’s disease, from Marfan syndrome to multiple sclerosis.  In collaboration with colleagues at Monash University, Melbourne Australia, we are also focussing on designing therapeutic strategies for much more prevalent human disorders, including asthma, diabetes and the highly relevant and topical COVID pandemic. If we can successfully develop antisense oligomers capable of increasing our COVID resistance or conferring anti-inflammatory properties where appropriate, massive demand will require a restructuring of oligomer scales of production and cost.

Keywords Antisense oligomers; Genetic Disease; Duchenne muscular dystrophy; Exon skipping; Isoform Switching, RAGE

Read more about Steve Wilton:
Professor Steve Wilton is the Director of the Centre for Molecular Medicine and Therapeutics at Murdoch University and the Perron Institute for Neurological and Translational Science (QE II Medical Centre, Western Australia).  He has positioned the Molecular Therapy Laboratory to become a pipeline of future drug development to treat many inherited and acquired diseases. The MTL is currently investigating treatments for more than 50 diseases, several of which are being undertaken with national and international collaborators.  He is committed to developing new therapeutics for many different conditions through “Therapeutic Alternative Splicing”, using antisense compounds to redirect gene expression as required for each condition: targeted exon skipping to bypass disease-causing mutations, promote retention of exons lost through splice motif mutations or disrupt expression of toxic gene products.

The US FDA granted accelerated approval for the first dystrophin exon-skipping drug (Eteplirsen) in September 2016, a second drug Golodirsen was approved in 2019 and Casimersen received accelerated approval in February 2021 as treatments for Duchenne muscular dystrophy.  With longtime colleague Prof Sue Fletcher, their work has been recognized with the 2012 Western Australian Innovator of the Year award, the 2013 Australian Museum Eureka Award for Translational Medicine, the 2014 LabGear Australia Discovery award, a finalist in the 2016 Western Australian of the Year (Professions Category) and a finalist for the Prime Minister’s Award for Science (2019).