Poster presentations
i. Basic science
ii. Translational
iii. Clinical
| Friday, November 8, 2024 |
| 10:30 AM - 11:00 AM |
| Leighton Hall, John Niland Scientia Building |
Dr Steven Eamegdool
Children's Medical Research Institute
P101 Steps to resolution of variants of uncertain significance in RPE65
Abstract
P101 Steps to resolution of variants of uncertain significance in RPE65
Background: Early-onset severe retinal dystrophy and Leber congenital amaurosis lead to marked visual impairment in childhood and progressive visual loss. These conditions may be caused by variants in the RPE65 gene, which encodes an isomerisation enzyme found in retinal pigment epithelial cells (RPE). Affected patients with bi-allelic, pathological variants in this gene may benefit from the sight-saving RPE65 gene therapy, voretigene neparvovec-rzyl (Luxturna, Novartis). However, approximately a third of the variants reported in RPE65 in the international variant database, ClinVar, are classified as variants of uncertain significance (VUS), rendering patients ineligible for therapy. Functional assessment of these variants is crucial for therapy eligibility. In this study we are investigating biomarkers, including gene/protein expression and high pressure liquid chromatography (HPLC) in induced pluripotent stem cell-derived RPE (iRPE), to aid variant interpretation and therapy eligibility.
Methods: CRISPR-Cas9 gene editing technology was used on normal iPSCs to introduce missense variants in RPE65. Cells containing normal or missense variants in RPE65 were expanded and differentiated. RPE65 transcript and protein expression levels were validated through RT-qPCR and western blot respectively. In addition, steps were undertaken to optimise the HPLC methodology using retinoid standards.
Results: We have found that iRPE containing RPE65 missense variants may lead to reduced expression of RPE65 both at the RNA and protein level, compared to normal control. We have also optimised the HPLC methodology for accurately detecting the various retinoids associated with RPE65 function.
Conclusion: We have optimised methods to detect functional differences in isomerisation capability of normal RPE65 compared with RPE65 containing missense variants. This is a crucial stepping stone in reclassifying RPE65 variants in patients, enabling them an opportunity for access to sight-saving gene therapy.
Background: Early-onset severe retinal dystrophy and Leber congenital amaurosis lead to marked visual impairment in childhood and progressive visual loss. These conditions may be caused by variants in the RPE65 gene, which encodes an isomerisation enzyme found in retinal pigment epithelial cells (RPE). Affected patients with bi-allelic, pathological variants in this gene may benefit from the sight-saving RPE65 gene therapy, voretigene neparvovec-rzyl (Luxturna, Novartis). However, approximately a third of the variants reported in RPE65 in the international variant database, ClinVar, are classified as variants of uncertain significance (VUS), rendering patients ineligible for therapy. Functional assessment of these variants is crucial for therapy eligibility. In this study we are investigating biomarkers, including gene/protein expression and high pressure liquid chromatography (HPLC) in induced pluripotent stem cell-derived RPE (iRPE), to aid variant interpretation and therapy eligibility.
Methods: CRISPR-Cas9 gene editing technology was used on normal iPSCs to introduce missense variants in RPE65. Cells containing normal or missense variants in RPE65 were expanded and differentiated. RPE65 transcript and protein expression levels were validated through RT-qPCR and western blot respectively. In addition, steps were undertaken to optimise the HPLC methodology using retinoid standards.
Results: We have found that iRPE containing RPE65 missense variants may lead to reduced expression of RPE65 both at the RNA and protein level, compared to normal control. We have also optimised the HPLC methodology for accurately detecting the various retinoids associated with RPE65 function.
Conclusion: We have optimised methods to detect functional differences in isomerisation capability of normal RPE65 compared with RPE65 containing missense variants. This is a crucial stepping stone in reclassifying RPE65 variants in patients, enabling them an opportunity for access to sight-saving gene therapy.
Biography
Steven is a postdoctoral research fellow in the Eye Genetics Research Unit, Children’s Medical Research Institute (CMRI), and received his PhD in 2015 from the lab of Prof Tailoi Chan-ling, University of Sydney. His area of research was in neural stem cells and nanoparticles. Following this, he worked as a post-doctoral researcher at the Save Sight Institute, under A/Prof Michele Madigan, focusing on the biology of retinal pigment epithelial cells and their association with the pathology of aged macular degeneration. In 2017, he then moved to Prof Robyn Jamieson's lab, where he developed a special interest in understanding the impact of genetic variants in functional assays in ocular diseases, and development of novel therapies in the retinal dystrophies. His work has contributed to development of two novel therapy approaches from in the laboratory with related patent applications and publications under review or in progress.
Miss Reeva Nadkar
Phd Candidate
Children's Medical Research Institute
P102 Genetic editing applications for variant classification and therapy development for cone-rod dystrophies
Abstract
P102 Genetic editing applications for variant classification and therapy development for cone-rod dystrophies
Background & Aims: Inherited retinal dystrophies (IRDs) affect around 1 in 1,000 individuals and frequently result in progressive degeneration of retinal cells, and eventually blindness. Variants occurring in genes and proteins, such as ABCA4, PROM1 and RPGR, affect photoreceptors and retinal pigment epithelium (RPE) cells and cause IRDs, such as Cone-Rod Dystrophies (CRD). Two major concerns in CRD include classification of variants of uncertain significance (VUS) and current unavailability of therapies for recurrent variants. The aim of this study is to characterise and reclassify VUS in CRD-causing genes and to design novel therapy approaches for recurrent variants. Both aims will be achieved using genetic editing in vitro with reference to the American College of Medical Genetics and Genomics (ACMG) criteria for variant analysis.
Methods: For VUS investigation, known pathogenic, benign and uncertain variants were introduced into control human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas-9 Homology-Directed Repair (HDR) and were then differentiated into retinal tissues, with a particular focus on the RPE. For therapy development, a prime editing strategy was designed and investigated to assess correction efficiency. Additional controls and patient-derived hiPSCs were used for comparisons in both aims.
Results: Using CRISPR/Cas-9 HDR, six hiPSC lines were created each containing a VUS, pathogenic or benign variant. Microarray and sequencing studies confirmed no off-targeting events in these lines prior to use in subsequent investigation. Multiple patient-derived hiPSCs differentiated into RPE revealed reduced subject protein expression when compared to control. CRISPR/Cas-9 HDR in HEK293Ts successfully introduced a recurrent variant for therapy development. Prime editing plasmids were designed to test correction efficiency in HEK293T disease model in preparation for application in patient-derived hiPSCs.
Conclusions: Genetic editing approaches are useful for reclassification of VUS and novel therapy development targeting recurrent CRD-variants. Optimisation of this workflow will be valuable and applicable for other IRDs.
Background & Aims: Inherited retinal dystrophies (IRDs) affect around 1 in 1,000 individuals and frequently result in progressive degeneration of retinal cells, and eventually blindness. Variants occurring in genes and proteins, such as ABCA4, PROM1 and RPGR, affect photoreceptors and retinal pigment epithelium (RPE) cells and cause IRDs, such as Cone-Rod Dystrophies (CRD). Two major concerns in CRD include classification of variants of uncertain significance (VUS) and current unavailability of therapies for recurrent variants. The aim of this study is to characterise and reclassify VUS in CRD-causing genes and to design novel therapy approaches for recurrent variants. Both aims will be achieved using genetic editing in vitro with reference to the American College of Medical Genetics and Genomics (ACMG) criteria for variant analysis.
Methods: For VUS investigation, known pathogenic, benign and uncertain variants were introduced into control human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas-9 Homology-Directed Repair (HDR) and were then differentiated into retinal tissues, with a particular focus on the RPE. For therapy development, a prime editing strategy was designed and investigated to assess correction efficiency. Additional controls and patient-derived hiPSCs were used for comparisons in both aims.
Results: Using CRISPR/Cas-9 HDR, six hiPSC lines were created each containing a VUS, pathogenic or benign variant. Microarray and sequencing studies confirmed no off-targeting events in these lines prior to use in subsequent investigation. Multiple patient-derived hiPSCs differentiated into RPE revealed reduced subject protein expression when compared to control. CRISPR/Cas-9 HDR in HEK293Ts successfully introduced a recurrent variant for therapy development. Prime editing plasmids were designed to test correction efficiency in HEK293T disease model in preparation for application in patient-derived hiPSCs.
Conclusions: Genetic editing approaches are useful for reclassification of VUS and novel therapy development targeting recurrent CRD-variants. Optimisation of this workflow will be valuable and applicable for other IRDs.
Biography
Reeva is a second year PhD Candidate from the Faculty of Health and Medicine at The University of Sydney. She is working in the Eye Genetics Research Unit at the Children's Medical Research Institute (CMRI). Reeva's project involves the investigation of Inherited Retinal Dystrophy-causing genes and their implications in clinical management.
Dr Carolyn Arico
The University of Sydney
P103 Estimating population prevalence of rare diseases in children using administrative health data
Abstract
P103 Estimating population prevalence of rare diseases in children using administrative health data
Background: Although around 8% of the population is estimated to have a rare disease, a major challenge is accurately identifying these individuals in the health system. The true prevalence in Australia is currently unknown. While Orphanet provides a classification system for rare diseases, this has not been applied in NSW. We aimed to apply Orphanet classification to NSW administrative health data to investigate the prevalence of paediatric rare diseases.
Methods: We conducted an NSW-wide population-based study of all children <18 years admitted to hospital who received a rare disease code between 2010-2019. Data were ascertained from the NSW Admitted Patient Data Collection where diagnoses associated with each admission are coded using the International Classification of Disease version 10-Australian Modification (ICD10-AM). We mapped 6,207 ORPHA codes to 3,230 ICD10-AM codes using the 2023 Orphanet Masterfile and Walker et al. (2017) resource to classify rare diseases in our data, and aggregated these to 31 primary medical categories. Overall frequency and rate in 2018 by major categories and by sex and age-group were determined.
Results: 300,127/ 1,605,491 (18.7%) children admitted to a NSW hospital in 2010-2019 had a rare disease code, 55% were male with mean age of 5.1 years (SD 5.7 years). The three most common categories were ‘rare neurologic diseases’ (19.5%), ‘rare developmental defects during embryogenesis’ (19.3%) and ‘rare respiratory diseases’ (9.7%). Prevalence of rare diseases requiring hospitalisation in 2018 was 2.3%; rates ranging between 0.001-0.778%.
Conclusion: We have developed a contemporary coding set to map Orphanet codes to ICD10-AM and demonstrated its application to enable use of administrative health data to calculate population prevalence. With linked data, this approach provides the potential to track cross-sectoral outcomes of rare diseases, critical for planning appropriate models of care for these collectively common and impactful conditions, and assess impact of precision medicine initiatives.
Background: Although around 8% of the population is estimated to have a rare disease, a major challenge is accurately identifying these individuals in the health system. The true prevalence in Australia is currently unknown. While Orphanet provides a classification system for rare diseases, this has not been applied in NSW. We aimed to apply Orphanet classification to NSW administrative health data to investigate the prevalence of paediatric rare diseases.
Methods: We conducted an NSW-wide population-based study of all children <18 years admitted to hospital who received a rare disease code between 2010-2019. Data were ascertained from the NSW Admitted Patient Data Collection where diagnoses associated with each admission are coded using the International Classification of Disease version 10-Australian Modification (ICD10-AM). We mapped 6,207 ORPHA codes to 3,230 ICD10-AM codes using the 2023 Orphanet Masterfile and Walker et al. (2017) resource to classify rare diseases in our data, and aggregated these to 31 primary medical categories. Overall frequency and rate in 2018 by major categories and by sex and age-group were determined.
Results: 300,127/ 1,605,491 (18.7%) children admitted to a NSW hospital in 2010-2019 had a rare disease code, 55% were male with mean age of 5.1 years (SD 5.7 years). The three most common categories were ‘rare neurologic diseases’ (19.5%), ‘rare developmental defects during embryogenesis’ (19.3%) and ‘rare respiratory diseases’ (9.7%). Prevalence of rare diseases requiring hospitalisation in 2018 was 2.3%; rates ranging between 0.001-0.778%.
Conclusion: We have developed a contemporary coding set to map Orphanet codes to ICD10-AM and demonstrated its application to enable use of administrative health data to calculate population prevalence. With linked data, this approach provides the potential to track cross-sectoral outcomes of rare diseases, critical for planning appropriate models of care for these collectively common and impactful conditions, and assess impact of precision medicine initiatives.
Biography
Carolyn is a Senior Research Officer working on the rare diseases project in the Child Population and Translational Health Research team at the University of Sydney. She has 5 years of experience working in data linkage infrastructure, having previously worked at the Centre for Health Record Linkage and the Sax Institute. She has a PhD in Behavioural Neuroscience and recently completed a Master of Public Health at the University of Sydney.
Dr Carolyn Arico
The University of Sydney
P104 Systematic review of population-based studies investigating rare diseases in children
Abstract
P104 Systematic review of population-based studies investigating rare diseases in children
Background: Rare diseases are estimated to affect 6-8% of the world’s population. Few population-based studies examine rare diseases as a collective, instead focusing on specific rare diseases. The aim of this study was to systematically review, identify and evaluate the methods utilised and prevalence estimated by population-based studies on rare diseases in children.
Methods: A search of MEDLINE via Ovid was conducted from January 2014 to June 2024 for population-based studies of rare diseases. Titles and abstracts were screened for population-based studies, published in English, focused on more than one type and/or category of rare disease. Supplementary reference/citation search was also conducted to identify further relevant studies. Two reviewers screened full-text articles for inclusion and performed data extraction. Information on study setting, time-period, data sources, study population (paediatric), classification, measures and main findings were extracted.
Results: 15 population-based studies met the inclusion criteria. Studies identified rare diseases using two main methods, via: 1) a population registry of patients with rare diseases, or 2) administrative health data sources such as hospital admission or mortality data using ICD10 or Orphanet diagnostic codes to identify patients with rare diseases. Studies examined different measures of rare diseases including population prevalence, proportion of the study cohort affected by a rare disease, mortality and hospital length of stay. Of the three studies that examined prevalence of rare diseases as a group, prevalence ranged between 1.5-2.3% of the population.
Conclusion: There are few population-based studies examining prevalence of rare diseases as a group, and of these, different sources, measures and outcomes of rare disease were assessed. Standardised classification of rare diseases using administrative health data would facilitate more population-based studies to enable investigators to examine real-world epidemiological data for rare diseases.
Background: Rare diseases are estimated to affect 6-8% of the world’s population. Few population-based studies examine rare diseases as a collective, instead focusing on specific rare diseases. The aim of this study was to systematically review, identify and evaluate the methods utilised and prevalence estimated by population-based studies on rare diseases in children.
Methods: A search of MEDLINE via Ovid was conducted from January 2014 to June 2024 for population-based studies of rare diseases. Titles and abstracts were screened for population-based studies, published in English, focused on more than one type and/or category of rare disease. Supplementary reference/citation search was also conducted to identify further relevant studies. Two reviewers screened full-text articles for inclusion and performed data extraction. Information on study setting, time-period, data sources, study population (paediatric), classification, measures and main findings were extracted.
Results: 15 population-based studies met the inclusion criteria. Studies identified rare diseases using two main methods, via: 1) a population registry of patients with rare diseases, or 2) administrative health data sources such as hospital admission or mortality data using ICD10 or Orphanet diagnostic codes to identify patients with rare diseases. Studies examined different measures of rare diseases including population prevalence, proportion of the study cohort affected by a rare disease, mortality and hospital length of stay. Of the three studies that examined prevalence of rare diseases as a group, prevalence ranged between 1.5-2.3% of the population.
Conclusion: There are few population-based studies examining prevalence of rare diseases as a group, and of these, different sources, measures and outcomes of rare disease were assessed. Standardised classification of rare diseases using administrative health data would facilitate more population-based studies to enable investigators to examine real-world epidemiological data for rare diseases.
Biography
Carolyn is a Senior Research Officer working on the rare diseases project in the Child Population and Translational Health Research team at the University of Sydney. She has 5 years of experience working in data linkage infrastructure, having previously worked at the Centre for Health Record Linkage and the Sax Institute. She has a PhD in Behavioural Neuroscience and recently completed a Master of Public Health at the University of Sydney.
Miss Elizabeth Kalotay
UNSW Sydney
P105 AAV-mediated DARS1 gene replacement in preclinical models of the leukodystrophy HBSL
Abstract
P105 AAV-mediated DARS1 gene replacement in preclinical models of the leukodystrophy HBSL
Leukodystrophies are a group of heritable diseases affecting the development and maintenance of white matter within the central nervous system (CNS). Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is a currently untreatable leukodystrophy that leads to severe physical disability, neurological abnormalities, and shortened life expectancy. HBSL is caused by biallelic loss-of-function mutations in the DARS1 gene, which encodes the cytosolic aspartyl-tRNA synthetase; an enzyme essential for protein synthesis. Here, we have developed and tested adeno-associated virus (AAV)-mediated DARS1 gene replacement to establish proof-of-concept for the treatment of HBSL.
We have generated two novel mouse models of DARS1 deficiency within the CNS, through conditional knockout of Dars1 in oligodendrocytes (Dars1OligoKO) and neurons (Dars1NeuroKO) of C57Bl/6J mice, using the Cre-loxP system. Concurrently, we have developed AAV vectors for targeted delivery of functional human DARS1. To maximise CNS transduction in this proof-of-principle study, we have selected the AAV.PHPeB capsid serotype. Our AAV gene expression cassette includes a codon-optimised and cytosine-phosphate-guanine (CpG) motif-free DARS1 coding sequence to promote strong, safe and long-term transgene expression.
Both Dars1OligoKO and Dars1NeuroKO mice exhibit pronounced pathological phenotypes that resemble key aspects of HBSL, including neurodegeneration, motor dysfunction, and reduced survival. Systemic delivery of our optimised AAV.DARS1 vector has shown significant efficacy in rescuing the phenotype of our HBSL model mice.
Our results provide proof-of-concept for an AAV-based DARS1 gene replacement therapy for HBSL. More broadly, our targeted gene delivery and disease modelling approaches will guide the development of AAV-mediated gene therapies for other leukodystrophies and monogenic diseases of the CNS.
Leukodystrophies are a group of heritable diseases affecting the development and maintenance of white matter within the central nervous system (CNS). Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is a currently untreatable leukodystrophy that leads to severe physical disability, neurological abnormalities, and shortened life expectancy. HBSL is caused by biallelic loss-of-function mutations in the DARS1 gene, which encodes the cytosolic aspartyl-tRNA synthetase; an enzyme essential for protein synthesis. Here, we have developed and tested adeno-associated virus (AAV)-mediated DARS1 gene replacement to establish proof-of-concept for the treatment of HBSL.
We have generated two novel mouse models of DARS1 deficiency within the CNS, through conditional knockout of Dars1 in oligodendrocytes (Dars1OligoKO) and neurons (Dars1NeuroKO) of C57Bl/6J mice, using the Cre-loxP system. Concurrently, we have developed AAV vectors for targeted delivery of functional human DARS1. To maximise CNS transduction in this proof-of-principle study, we have selected the AAV.PHPeB capsid serotype. Our AAV gene expression cassette includes a codon-optimised and cytosine-phosphate-guanine (CpG) motif-free DARS1 coding sequence to promote strong, safe and long-term transgene expression.
Both Dars1OligoKO and Dars1NeuroKO mice exhibit pronounced pathological phenotypes that resemble key aspects of HBSL, including neurodegeneration, motor dysfunction, and reduced survival. Systemic delivery of our optimised AAV.DARS1 vector has shown significant efficacy in rescuing the phenotype of our HBSL model mice.
Our results provide proof-of-concept for an AAV-based DARS1 gene replacement therapy for HBSL. More broadly, our targeted gene delivery and disease modelling approaches will guide the development of AAV-mediated gene therapies for other leukodystrophies and monogenic diseases of the CNS.
Biography
Elizabeth Kalotay is a PhD student within the CNS Gene Therapy group in the Translational Neuroscience Facility, within the School of Biomedical Sciences at UNSW. Prior to commencing her PhD, she completed her undergraduate degree in Advanced Science at UNSW, with majors in psychology and neuroscience. In 2020, she was awarded the University Medal in Neuroscience, and received the Paxinos and Watson award for best performance in the Neuroscience Honours program. During her PhD candidature, Elizabeth has published 4 papers (including 2 first author publications), and has presented her research at both national and international conferences.
Dr. Froehlich is a neuroscientist with a broad background in genetics, neuroscience, and molecular cell biology. At UNSW Sydney, he leads the CNS Gene Therapy group, which is part of the Translational Neuroscience Facility in the School of Biomedical Sciences. Dr. Froehlich has >14 years of experience in translational neuroscience including generation and characterisation of mouse models for nervous system disorders and injuries, and the development of targeted gene therapies for neurological diseases. He has published >20 peer-reviewed journal articles in the fields of neuroscience and gene therapy (7 first and 5 last author publications) with more than 2400 citations (FWCI 2.15). Dr. Froehlich is an Editorial Board member of the Rare Disease and Orphan Drugs Journal (RDODJ) and Frontiers in Cellular Neuroscience. He also engages with the non-scientific community in his roles as vice president and scientific advisor of Leukodystrophy Australia, and scientific advisor to the Mission Massimo Foundation.
Dr Dominik Froehlich
UNSW Sydney
Co-presenter - P105
Biography
Ms Puja Thapa
The University of Sydney
P106 Preclinical assessment of gene therapy for RARS2-related early onset epileptic encephalopathy
Abstract
P106 Preclinical assessment of gene therapy for RARS2-related early onset epileptic encephalopathy
Background: Developmental and epileptic encephalopathy (DEE) caused by biallelic RARS2 pathogenic variants is a rare neurological disorder that impairs cognitive and motor functions. RARS2 encodes the mitochondrial arginyl-tRNA synthetase protein which is essential for mitochondrial protein synthesis. Variants in RARS2 disrupt normal brain development, leading to early-onset epileptic encephalopathy where symptoms typically manifest in infancy, characterised by drug-resistant epilepsy, developmental delay, and profound intellectual disability. Understanding the molecular and cellular mechanisms underlying RARS2-related DEE remains challenging due to the scarcity of brain tissue samples and suitable animal models. In this project, we utilise human cortical brain organoids derived from induced pluripotent stem cells (iPSCs) to study RARS2-related DEE at the cellular and molecular level. Importantly we aim to establish proof of concept for AAV-RARS2 gene therapy as a potential therapeutic approach for this disease.
Methods: We used homology-directed repair CRISPR gene editing to generate an iPSC line with a RARS2 pathogenic variant (c.392T>G; p.(Phe131Cys)). These iPSCs were differentiated into dorsal cortical organoids to model RARS2-related DEE. The cortical organoids were cultured in suspension for several months to develop cortical layers and complex neuronal networks. An electrophysiology-relevant medium was introduced at 100 days in vitro to better support synaptic activity and neuronal function.
Results: Multi-electrode array recordings were performed in day 150 organoids revealing that RARS2-variant organoids exhibited higher neural activity compared to isogenic controls. Preliminary validation of the RARS2 gene therapy construct demonstrated an increase in RARS2 gene expression and protein.
Conclusion: These data demonstrated that brain organoids are suitable model systems to study neurodevelopmental disorders providing novel insights into network functionality in disease. Furthermore, this project will provide a pipeline for developing gene therapies for other mitochondrial disorders.
Background: Developmental and epileptic encephalopathy (DEE) caused by biallelic RARS2 pathogenic variants is a rare neurological disorder that impairs cognitive and motor functions. RARS2 encodes the mitochondrial arginyl-tRNA synthetase protein which is essential for mitochondrial protein synthesis. Variants in RARS2 disrupt normal brain development, leading to early-onset epileptic encephalopathy where symptoms typically manifest in infancy, characterised by drug-resistant epilepsy, developmental delay, and profound intellectual disability. Understanding the molecular and cellular mechanisms underlying RARS2-related DEE remains challenging due to the scarcity of brain tissue samples and suitable animal models. In this project, we utilise human cortical brain organoids derived from induced pluripotent stem cells (iPSCs) to study RARS2-related DEE at the cellular and molecular level. Importantly we aim to establish proof of concept for AAV-RARS2 gene therapy as a potential therapeutic approach for this disease.
Methods: We used homology-directed repair CRISPR gene editing to generate an iPSC line with a RARS2 pathogenic variant (c.392T>G; p.(Phe131Cys)). These iPSCs were differentiated into dorsal cortical organoids to model RARS2-related DEE. The cortical organoids were cultured in suspension for several months to develop cortical layers and complex neuronal networks. An electrophysiology-relevant medium was introduced at 100 days in vitro to better support synaptic activity and neuronal function.
Results: Multi-electrode array recordings were performed in day 150 organoids revealing that RARS2-variant organoids exhibited higher neural activity compared to isogenic controls. Preliminary validation of the RARS2 gene therapy construct demonstrated an increase in RARS2 gene expression and protein.
Conclusion: These data demonstrated that brain organoids are suitable model systems to study neurodevelopmental disorders providing novel insights into network functionality in disease. Furthermore, this project will provide a pipeline for developing gene therapies for other mitochondrial disorders.
Biography
Puja Thapa is a PhD Candidate in the Molecular Neurobiology Research Laboratory at Kid's Research, Sydney Children's Hospital Network. She is currently working on the preclinical assessment of gene therapy for RARS2-related early-onset epileptic encephalopathy. Puja holds a Bachelor of Science degree with honours from the University of Sydney.
Dr Alan Ma
Clinical Geneticist
The University of Sydney, Sydney Children's Hospitals Network
P107 PRECISE – Co-designing a genomics and precision medicine program for primary care
Abstract
P107 PRECISE – Co-designing a genomics and precision medicine program for primary care
Background: Precision medicine and the growing demand for genomics in all areas of medicine has led to massive challenges in service provision, education and upskilling for non-genetics healthcare professionals. This is particularly an issue in primary care, where genomics and precision medicine is increasingly seen as standard healthcare. GPs are able to order genomic tests such as in non-invasive prenatal screening, reproductive carrier testing, and are increasingly seeing patients with genetic conditions. However, there has been relatively slow uptake of genomics in primary care, with practitioners reporting inadequate capacity, training, and support to provide genomic care to patients.
The PRECISE (Practitioner Readiness, Education and Capabilities, with Implementation Science Evaluation) Project is a MRFF funded research program that will build capacity in genomics for primary care practitioners and evidence-based strategies for further implementation into the primary health care sector.
Methods: This project brings together a multidisciplinary team comprising of consumers, primary healthcare, academia, industry, decision makers and genetics services. It will utilise implementation science frameworks, as well as principles of education co-design, and program evaluation with stakeholders and consumers. This project aims to both co-design education resources and identify strategies to improve capacity within the primary healthcare sector. This is with an implementation science approach incorporating the Knowledge to Action cycle.
Results: Our initial scoping review has already identified key attitudes, needs, and gaps in genomic education in the sector. This will inform our co-design stage, to produce resources to support primary care practitioners in applying genomics, as well as knowledge on strategies to improve clinical capacity in the sector.
Conclusion: The PRECISE team is a unique and collaborative approach, bringing a diverse team of consumers, clinical genetics, postgraduate education, implementation science and evaluation, public health, primary care, industry and policy with the aim to build genomics capabilities in this sector.
Background: Precision medicine and the growing demand for genomics in all areas of medicine has led to massive challenges in service provision, education and upskilling for non-genetics healthcare professionals. This is particularly an issue in primary care, where genomics and precision medicine is increasingly seen as standard healthcare. GPs are able to order genomic tests such as in non-invasive prenatal screening, reproductive carrier testing, and are increasingly seeing patients with genetic conditions. However, there has been relatively slow uptake of genomics in primary care, with practitioners reporting inadequate capacity, training, and support to provide genomic care to patients.
The PRECISE (Practitioner Readiness, Education and Capabilities, with Implementation Science Evaluation) Project is a MRFF funded research program that will build capacity in genomics for primary care practitioners and evidence-based strategies for further implementation into the primary health care sector.
Methods: This project brings together a multidisciplinary team comprising of consumers, primary healthcare, academia, industry, decision makers and genetics services. It will utilise implementation science frameworks, as well as principles of education co-design, and program evaluation with stakeholders and consumers. This project aims to both co-design education resources and identify strategies to improve capacity within the primary healthcare sector. This is with an implementation science approach incorporating the Knowledge to Action cycle.
Results: Our initial scoping review has already identified key attitudes, needs, and gaps in genomic education in the sector. This will inform our co-design stage, to produce resources to support primary care practitioners in applying genomics, as well as knowledge on strategies to improve clinical capacity in the sector.
Conclusion: The PRECISE team is a unique and collaborative approach, bringing a diverse team of consumers, clinical genetics, postgraduate education, implementation science and evaluation, public health, primary care, industry and policy with the aim to build genomics capabilities in this sector.
Biography
Dr Alan Ma is an early career clinician researcher. He is a clinical geneticist at SCHN, and senior lecturer with the specialty of genomic medicine, University of Sydney. He is the lead investigator for PRECISE: a MRFF funded project for enabling genomics and precision medicine in primary care. His research interest is in implementation science, and the health services translation required to implement genomics into the healthcare system.
Miss Sarah Shin
Sydney Children’s Hospitals Network
P108 Newborn Screening for Duchenne muscular dystrophy: Development of a scoping study
Abstract
P108 Newborn Screening for Duchenne muscular dystrophy: Development of a scoping study
Duchenne muscular dystrophy (DMD) affects approximately 1 in 5000 live male births worldwide and is characterised by progressive muscle weakness and atrophy. Several dystrophin restoration therapies have been developed and serve as a promising therapeutic approach for DMD, however potential benefits of earlier treatment or intervention prior to disease onset are not well understood. Newborn Genomic Sequencing in Screening: Therapy Ready and Information for Life (TRAIL) TRAIL study has set out to investigate the potential use of genomic technologies to expand newborn screening (NBS) capabilities. The study aims to conduct the first DMD NBS scoping study in New South Wales (NSW) and the Australian Capital Territory (ACT) to evaluate the feasibility, acceptability, and benefit of screening newborns for DMD. The scoping study will run for 12-months, screening 100,000 newborns across NSW and ACT using dried blood spot (DBS) cards. The first-tier screen will measure muscle-specific creatine kinase isoform (CK-MM) concentrations. Rapid aneuploidy detection by QF-PCR will be performed on the cards with raised CK-MM levels to identify males. Third-tier screen will identify pathogenic variants in the DMD gene using a massively parallel sequencing capture panel on male screen positive cards. Newborns with pathogenic DMD variants will be referred for diagnostic testing and the model of care will be assessed for newborns diagnosed through the scoping study. The study findings will help inform future scoping studies and model of care approaches for other genetic conditions in the trajectory of genomic NBS and implementation into health practice and policy.
Duchenne muscular dystrophy (DMD) affects approximately 1 in 5000 live male births worldwide and is characterised by progressive muscle weakness and atrophy. Several dystrophin restoration therapies have been developed and serve as a promising therapeutic approach for DMD, however potential benefits of earlier treatment or intervention prior to disease onset are not well understood. Newborn Genomic Sequencing in Screening: Therapy Ready and Information for Life (TRAIL) TRAIL study has set out to investigate the potential use of genomic technologies to expand newborn screening (NBS) capabilities. The study aims to conduct the first DMD NBS scoping study in New South Wales (NSW) and the Australian Capital Territory (ACT) to evaluate the feasibility, acceptability, and benefit of screening newborns for DMD. The scoping study will run for 12-months, screening 100,000 newborns across NSW and ACT using dried blood spot (DBS) cards. The first-tier screen will measure muscle-specific creatine kinase isoform (CK-MM) concentrations. Rapid aneuploidy detection by QF-PCR will be performed on the cards with raised CK-MM levels to identify males. Third-tier screen will identify pathogenic variants in the DMD gene using a massively parallel sequencing capture panel on male screen positive cards. Newborns with pathogenic DMD variants will be referred for diagnostic testing and the model of care will be assessed for newborns diagnosed through the scoping study. The study findings will help inform future scoping studies and model of care approaches for other genetic conditions in the trajectory of genomic NBS and implementation into health practice and policy.
Biography
Sarah Shin is a Research Assistant involved in the Newborn Gen Seq TRAIL study at the Sydney Children's Hospital Network.
Mr Patrick Nay
ZERO Drug Access Navigator / Pharmacist
Kids Cancer Centre, Sydney Children's Hospital
P109 From recommendation to access: Improving procurement of precision-guided treatments with implementation science
Abstract
P109 From recommendation to access: Improving procurement of precision-guided treatments with implementation science
Background: ProCure is a novel online, interactive paediatric oncology medicines access database co-designed with healthcare providers (n=17, paediatric oncologists, nurse consultants and allied health professionals) and developed using implementation science methods to streamline the application process for accessing targeted therapies. Following beta testing with end users (n=12, paediatric oncologists, pharmacists and scientists) in September 2023, ProCure was found to an acceptable and appropriate resource. These findings combined with its co-design indicate that ProCure is ready for implementation. This study aims to assess its implementation and effectiveness at paediatric centres nationally using a two-arm, parallel, randomised cluster trial with a type 3 effectiveness-implementation hybrid design.
Methods: A focus group will co-design an implementation support package (ISP) through consultation with clinicians from each paediatric centre. The ISP will be based on targeted, evidence-based strategies informed by the Expert Recommendations for Implementing Change tool, to mitigate barriers previously identified using the Consolidated Framework for Implementation Research. ProCure will be delivered to all sites, but only one trial arm will receive the ISP. ProCure’s implementation outcomes and clinical effectiveness will be measured through inter-arm and pre- and post-trial comparisons of data collected from surveys, health system audit data, web metrics, and interviews. These analyses will be conducted separately for high-risk and non-high-risk patients.
Results: Higher adoption and sustained implementation of ProCure in the intervention arm would indicate the ISP’s effectiveness. Increased confidence among clinicians, along with a reduction in the time required to complete and submit compassionate access applications would demonstrate ProCure’s effectiveness as a service intervention.
Conclusion: Outcomes from this implementation trial will provide further insight into clinicians’ compassionate access of precision-guided treatments recommended by precision medicine trials. ProCure’s role as an intervention to facilitate uptake of precision oncology recommendations in a clinical context will inform future international scale-up of ProCure.
Background: ProCure is a novel online, interactive paediatric oncology medicines access database co-designed with healthcare providers (n=17, paediatric oncologists, nurse consultants and allied health professionals) and developed using implementation science methods to streamline the application process for accessing targeted therapies. Following beta testing with end users (n=12, paediatric oncologists, pharmacists and scientists) in September 2023, ProCure was found to an acceptable and appropriate resource. These findings combined with its co-design indicate that ProCure is ready for implementation. This study aims to assess its implementation and effectiveness at paediatric centres nationally using a two-arm, parallel, randomised cluster trial with a type 3 effectiveness-implementation hybrid design.
Methods: A focus group will co-design an implementation support package (ISP) through consultation with clinicians from each paediatric centre. The ISP will be based on targeted, evidence-based strategies informed by the Expert Recommendations for Implementing Change tool, to mitigate barriers previously identified using the Consolidated Framework for Implementation Research. ProCure will be delivered to all sites, but only one trial arm will receive the ISP. ProCure’s implementation outcomes and clinical effectiveness will be measured through inter-arm and pre- and post-trial comparisons of data collected from surveys, health system audit data, web metrics, and interviews. These analyses will be conducted separately for high-risk and non-high-risk patients.
Results: Higher adoption and sustained implementation of ProCure in the intervention arm would indicate the ISP’s effectiveness. Increased confidence among clinicians, along with a reduction in the time required to complete and submit compassionate access applications would demonstrate ProCure’s effectiveness as a service intervention.
Conclusion: Outcomes from this implementation trial will provide further insight into clinicians’ compassionate access of precision-guided treatments recommended by precision medicine trials. ProCure’s role as an intervention to facilitate uptake of precision oncology recommendations in a clinical context will inform future international scale-up of ProCure.
Biography
Patrick is a clinical pharmacist with 7 years of experience in hospital pharmacy and research. As the Drug Access Navigator for the national ZERO Childhood Cancer Program, he is passionate about novel targeted therapies and implementing new initiatives. Patrick is also working to implement and maintain a precision oncology drug database called ‘ProCure’ for ZERO clinicians and pharmacists. His role involves navigating drug availability, and procurement to ensure every child receives the best possible care. He collaborates closely with pharmaceutical companies and multidisciplinary teams to secure life-saving medications and optimise patient management, ultimately enhancing therapeutic outcomes and quality of life.
Dr Suzanne Nevin
UNSW Sydney
P110 Co-developing positive psychology resources with caregivers of children with developmental epileptic encephalopathies
Abstract
P110 Co-developing positive psychology resources with caregivers of children with developmental epileptic encephalopathies
Developmental and epileptic encephalopathies (DEEs) are a group of life-threatening and life-limiting conditions characterised by early-onset intractable seizures, debilitating co-morbidities, and premature mortality. Extensive progress has been made in understanding DEE pathophysiology: over 400 genes have been reported, with a growing number of genetic identifiers still being discovered. However, advances in gene diagnostics have yet to translate into precision therapies for affected children and the psychosocial resources to support families are limited. This research used a mixed-methods, multi-perspective approach to investigate caregiver and family psychosocial experiences undergoing genetic testing for DEE, to co-develop a suite of tailored psychosocial supports.
Phase one involved a systematic literature review of the international literature to identify caregivers’ information needs and preferences to support clinical practice. In phase two, I conducted a mixed-methods interview study investigating caregivers' experiences undergoing genetic testing, to identify their psychosocial support needs, before, during and after their child’s genetic testing. In phase three I convened a priority-setting project, engaging caregivers and multidisciplinary healthcare professionals to codesign a suite of psychological resources using a person-based approach. Based on the findings of phase one to three, I developed ‘Finding a Way’, a suite of audio-visual psychological resources tailored to support psychosocial adjustment to a child’s genetic DEE diagnosis. In stage four, I conducted a mixed-methods study to evaluate the acceptability and emotional impact of ‘Finding a Way’ among an international cohort of caregivers of children with DEEs. Our qualitative and quantitative data provided evidence that suggested personalized positive psychology resources ('Finding a Way') can enhance caregiver emotional adaptation to their child's genetic DEE diagnosis.
This translational program of work delivered high-quality, innovative psychological resources tailored to address specific psychosocial challenges experienced among caregivers of children with genetic DEEs.
Developmental and epileptic encephalopathies (DEEs) are a group of life-threatening and life-limiting conditions characterised by early-onset intractable seizures, debilitating co-morbidities, and premature mortality. Extensive progress has been made in understanding DEE pathophysiology: over 400 genes have been reported, with a growing number of genetic identifiers still being discovered. However, advances in gene diagnostics have yet to translate into precision therapies for affected children and the psychosocial resources to support families are limited. This research used a mixed-methods, multi-perspective approach to investigate caregiver and family psychosocial experiences undergoing genetic testing for DEE, to co-develop a suite of tailored psychosocial supports.
Phase one involved a systematic literature review of the international literature to identify caregivers’ information needs and preferences to support clinical practice. In phase two, I conducted a mixed-methods interview study investigating caregivers' experiences undergoing genetic testing, to identify their psychosocial support needs, before, during and after their child’s genetic testing. In phase three I convened a priority-setting project, engaging caregivers and multidisciplinary healthcare professionals to codesign a suite of psychological resources using a person-based approach. Based on the findings of phase one to three, I developed ‘Finding a Way’, a suite of audio-visual psychological resources tailored to support psychosocial adjustment to a child’s genetic DEE diagnosis. In stage four, I conducted a mixed-methods study to evaluate the acceptability and emotional impact of ‘Finding a Way’ among an international cohort of caregivers of children with DEEs. Our qualitative and quantitative data provided evidence that suggested personalized positive psychology resources ('Finding a Way') can enhance caregiver emotional adaptation to their child's genetic DEE diagnosis.
This translational program of work delivered high-quality, innovative psychological resources tailored to address specific psychosocial challenges experienced among caregivers of children with genetic DEEs.
Biography
Dr Suzanne Nevin is a post-doctoral researcher and provisional psychologist at the Sydney Children's Hospital and the University of New South Wales. Her research is focussed on partnering with families of children living with rare neurodegenerative conditions and codesigning psychological and psychosocial resources tailored to promote positive emotional adaptation. She endeavours to connect with families and children with complex neurodevelopmental, and behavioural conditions using collaborative, and family-centred evidence-based approaches to design and deliver meaningful resources that can support families to live their best lives possible.
Dr Laszlo Irinyi
Senior Project Officer
NSW Health Pathology
P111 A pilot study of creating a long-term follow-up clinical registry and biorepository of patients undergoing cell and gene therapies
Abstract
P111 A pilot study of creating a long-term follow-up clinical registry and biorepository of patients undergoing cell and gene therapies
Background: Emerging treatments such as cell and gene therapies (CGT) offer new possibilities for treating challenging medical conditions with limited or no treatment options, from rare inherited disorders to certain forms of cancer. These therapies aim to achieve therapeutic effects through long-lasting or permanent changes in the human body. However, their interaction at the cellular and genetic levels carries risks, such as delayed adverse events and uncertain long-term benefits. Therefore, extended monitoring, or long-term follow-up (LTFU), is essential for safety, efficacy, treatment durability, understanding late-onset effects, regulatory compliance, future research, and ensuring patient well-being.
Methods: Our pilot will explore the collection and storage of data from multiple sources and biospecimens in the NSW Health Statewide Biobank, as well as the process of data linkage for long-term follow-up of patients receiving CGT products. Our platform will facilitate the extraction of data from medical records, including real-world data and patient-reported outcomes, and enable linkage with other existing databases. This process will be supported by transparent and consistent data governance practices.
Results: The study is in the planning phase and is actively seeking investigator-initiated clinical trials in CGT to support the development of a long-term follow-up database for CGT products. This pilot study would serve to demonstrate NSW’s ongoing leadership in gene and cell therapy, as well as emerging leadership capabilities in new registry models
Conclusion: Creating an accessible LTFU database and biorepository with data linkage for CGT products could enhance understanding of these therapies' safety and effectiveness over time, benefiting patients, clinicians, researchers, industry, and policymakers. Storing biospecimens in the NSW Health Statewide Biobank could also support research on under-studied diseases, like childhood dementia, and assist in monitoring these therapies.
Background: Emerging treatments such as cell and gene therapies (CGT) offer new possibilities for treating challenging medical conditions with limited or no treatment options, from rare inherited disorders to certain forms of cancer. These therapies aim to achieve therapeutic effects through long-lasting or permanent changes in the human body. However, their interaction at the cellular and genetic levels carries risks, such as delayed adverse events and uncertain long-term benefits. Therefore, extended monitoring, or long-term follow-up (LTFU), is essential for safety, efficacy, treatment durability, understanding late-onset effects, regulatory compliance, future research, and ensuring patient well-being.
Methods: Our pilot will explore the collection and storage of data from multiple sources and biospecimens in the NSW Health Statewide Biobank, as well as the process of data linkage for long-term follow-up of patients receiving CGT products. Our platform will facilitate the extraction of data from medical records, including real-world data and patient-reported outcomes, and enable linkage with other existing databases. This process will be supported by transparent and consistent data governance practices.
Results: The study is in the planning phase and is actively seeking investigator-initiated clinical trials in CGT to support the development of a long-term follow-up database for CGT products. This pilot study would serve to demonstrate NSW’s ongoing leadership in gene and cell therapy, as well as emerging leadership capabilities in new registry models
Conclusion: Creating an accessible LTFU database and biorepository with data linkage for CGT products could enhance understanding of these therapies' safety and effectiveness over time, benefiting patients, clinicians, researchers, industry, and policymakers. Storing biospecimens in the NSW Health Statewide Biobank could also support research on under-studied diseases, like childhood dementia, and assist in monitoring these therapies.
Biography
Miss Richa Chaluvadi
UNSW Sydney
P112 Gene therapy proof-of-concept for spastic paraplegia 56
Abstract
P112 Gene therapy proof-of-concept for spastic paraplegia 56
Hereditary spastic paraplegias (HSP) are a group of neurological disorders characterized by progressive lower limb weakness and spasticity; with a global prevalence of 3.6 in 1000 people. SPG56 is a subtype of HSP caused by autosomal recessive mutations of the CYP2U1 gene, which encodes CYP2U1, a cytochrome P450 hydroxylase predominantly found in cells of the central nervous system (CNS) and thymus. To date, no curative treatments exist. This study tests the efficacy of adeno-associated virus (AAV)-mediated CYP2U1 gene replacement in a Cyp2u1-/- knockout mouse model to establish the proof-of-concept for SPG56 gene therapy.
AAV9 was used for effective CNS transduction paired with either the CAG (AAV9.CAG.hCYP2U1) or EF1a promoter (AAV9.EF1a.hCYP2U1) for strong and moderate CYP2U1 expression, respectively. Cyp2u1-/- knockout mice were treated via intravenous or intracerebroventricular injections. Controls included untreated knockout, heterozygous and wild-type mice. Behavioural tests assessed motor function and learning, whilst blood samples were analysed for serum biomarker levels (CoQ9 and CoQ10). Post-mortem biochemical and histological analyses were performed to evaluate vector distribution and transgene expression.
First, the validity of the Cyp2u1-/- mouse strain as an SPG56 model was established. Serum CoQ9 and CoQ10 levels were elevated in knockout mice compared to wild-type and heterozygous controls, consistent with human SPG56 patients. Cyp2u1-/- mice showed sensorimotor gating deficits, indicated by reduced pre-pulse inhibition of the acoustic startle response, along with learning deficits on the y-maze and passive avoidance tests. Next, preliminary results of AAV-mediated CYP2U1 gene replacement showed significantly lower CoQ9 and CoQ10 levels and depicted trends towards normalisation of behavioural impairments.
This study provides the proof-of-concept for CYP2U1 gene replacement as an effective treatment for HSP SPG56, which paves the way for further development and clinical translation of this therapy – holding great promise to permanently cure this debilitating condition.
Hereditary spastic paraplegias (HSP) are a group of neurological disorders characterized by progressive lower limb weakness and spasticity; with a global prevalence of 3.6 in 1000 people. SPG56 is a subtype of HSP caused by autosomal recessive mutations of the CYP2U1 gene, which encodes CYP2U1, a cytochrome P450 hydroxylase predominantly found in cells of the central nervous system (CNS) and thymus. To date, no curative treatments exist. This study tests the efficacy of adeno-associated virus (AAV)-mediated CYP2U1 gene replacement in a Cyp2u1-/- knockout mouse model to establish the proof-of-concept for SPG56 gene therapy.
AAV9 was used for effective CNS transduction paired with either the CAG (AAV9.CAG.hCYP2U1) or EF1a promoter (AAV9.EF1a.hCYP2U1) for strong and moderate CYP2U1 expression, respectively. Cyp2u1-/- knockout mice were treated via intravenous or intracerebroventricular injections. Controls included untreated knockout, heterozygous and wild-type mice. Behavioural tests assessed motor function and learning, whilst blood samples were analysed for serum biomarker levels (CoQ9 and CoQ10). Post-mortem biochemical and histological analyses were performed to evaluate vector distribution and transgene expression.
First, the validity of the Cyp2u1-/- mouse strain as an SPG56 model was established. Serum CoQ9 and CoQ10 levels were elevated in knockout mice compared to wild-type and heterozygous controls, consistent with human SPG56 patients. Cyp2u1-/- mice showed sensorimotor gating deficits, indicated by reduced pre-pulse inhibition of the acoustic startle response, along with learning deficits on the y-maze and passive avoidance tests. Next, preliminary results of AAV-mediated CYP2U1 gene replacement showed significantly lower CoQ9 and CoQ10 levels and depicted trends towards normalisation of behavioural impairments.
This study provides the proof-of-concept for CYP2U1 gene replacement as an effective treatment for HSP SPG56, which paves the way for further development and clinical translation of this therapy – holding great promise to permanently cure this debilitating condition.
Biography
Richa Chaluvadi is a fourth-year medical student at the University of New South Wales, Sydney. In 2024, she completed her Bachelor of Science (Medicine) honours degree in the CNS Gene Therapy Group, which is integrated in the Translational Neuroscience Facility within the UNSW School of Biomedical Sciences. Her project characterised a novel mouse model for the hereditary spastic paraplegia SPG56 and tested the efficacy of AAV-mediated gene replacement therapy for this condition.
Dr. Froehlich leads the CNS Gene Therapy Group, which is part of the Translational Neuroscience Facility (TNF) at UNSW Sydney. He has >14 years of experience in translational neuroscience including generation and characterisation of mouse models for nervous system disorders and injuries, and the development of targeted gene therapies for neurological diseases. He has published >20 peer-reviewed journal articles in the fields of neuroscience and gene therapy (7 first and 5 last author publications) with more than 2400 citations (FWCI 2.15). Dr. Froehlich is an Editorial Board member of the Rare Disease and Orphan Drugs Journal (RDODJ) and Frontiers in Cellular Neuroscience. He also contributes to the wider community in his roles as vice president and scientific advisor of Leukodystrophy Australia, and scientific advisor to the Mission Massimo Foundation.
Dr Dominik Froehlich
UNSW Sydney
Co-presenter - P112
Biography
