AAV9-mediated SMN gene therapy rescues cardiac desmin but not lamin A/C and elastin dysregulation in Smn2B/- spinal muscular atrophy mice.

Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/- SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/- mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/- mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.

Enhanced expression of the human Survival motor neuron 1 gene from a codon-optimised cDNA transgene in vitro and in vivo.

Spinal muscular atrophy (SMA) is a neuromuscular disease particularly characterised by degeneration of ventral motor neurons. Survival motor neuron (SMN) 1 gene mutations cause SMA, and gene addition strategies to replace the faulty SMN1 copy are a therapeutic option. We have developed a novel, codon-optimised hSMN1 transgene and produced integration-proficient and integration-deficient lentiviral vectors with cytomegalovirus (CMV), human synapsin (hSYN) or human phosphoglycerate kinase (hPGK) promoters to determine the optimal expression cassette configuration. Integrating, CMV-driven and codon-optimised hSMN1 lentiviral vectors resulted in the highest production of functional SMN protein in vitro. Integration-deficient lentiviral vectors also led to significant expression of the optimised transgene and are expected to be safer than integrating vectors. Lentiviral delivery in culture led to activation of the DNA damage response, in particular elevating levels of phosphorylated ataxia telangiectasia mutated (pATM) and γH2AX, but the optimised hSMN1 transgene showed some protective effects. Neonatal delivery of adeno-associated viral vector (AAV9) vector encoding the optimised transgene to the Smn2B/- mouse model of SMA resulted in a significant increase of SMN protein levels in liver and spinal cord. This work shows the potential of a novel codon-optimised hSMN1 transgene as a therapeutic strategy for SMA.

Clinical applications of gene therapy for rare diseases: A review.

Rare diseases collectively exact a high toll on society due to their sheer number and overall prevalence. Their heterogeneity, diversity, and nature pose daunting clinical challenges for both management and treatment. In this review, we discuss recent advances in clinical applications of gene therapy for rare diseases, focusing on a variety of viral and non-viral strategies. The use of adeno-associated virus (AAV) vectors is discussed in the context of Luxturna, licenced for the treatment of RPE65 deficiency in the retinal epithelium. Imlygic, a herpes virus vector licenced for the treatment of refractory metastatic melanoma, will be an example of oncolytic vectors developed against rare cancers. Yescarta and Kymriah will showcase the use of retrovirus and lentivirus vectors in the autologous ex vivo production of chimeric antigen receptor T cells (CAR-T), licenced for the treatment of refractory leukaemias and lymphomas. Similar retroviral and lentiviral technology can be applied to autologous haematopoietic stem cells, exemplified by Strimvelis and Zynteglo, licenced treatments for adenosine deaminase-severe combined immunodeficiency (ADA-SCID) and ß-thalassaemia respectively. Antisense oligonucleotide technologies will be highlighted through Onpattro and Tegsedi, RNA interference drugs licenced for familial transthyretin (TTR) amyloidosis, and Spinraza, a splice-switching treatment for spinal muscular atrophy (SMA). An initial comparison of the effectiveness of AAV and oligonucleotide therapies in SMA is possible with Zolgensma, an AAV serotype 9 vector, and Spinraza. Through these examples of marketed gene therapies and gene cell therapies, we will discuss the expanding applications of such novel technologies to previously intractable rare diseases.

Systematic review and meta-analysis determining the benefits of in vivo genetic therapy in spinal muscular atrophy rodent models.

Spinal muscular atrophy (SMA) is a severe childhood neuromuscular disease for which two genetic therapies, Nusinersen (Spinraza, an antisense oligonucleotide), and AVXS-101 (Zolgensma, an adeno-associated viral vector of serotype 9 AAV9), have recently been approved. We investigated the pre-clinical development of SMA genetic therapies in rodent models and whether this can predict clinical efficacy. We have performed a systematic review of relevant publications and extracted median survival and details of experimental design. A random effects meta-analysis was used to estimate and compare efficacy. We stratified by experimental design (type of genetic therapy, mouse model, route and time of administration) and sought any evidence of publication bias. 51 publications were identified containing 155 individual comparisons, comprising 2573 animals in total. Genetic therapies prolonged survival in SMA mouse models by 3.23-fold (95% CI 2.75-3.79) compared to controls. Study design characteristics accounted for significant heterogeneity between studies and greatly affected observed median survival ratios. Some evidence of publication bias was found. These data are consistent with the extended average lifespan of Spinraza- and Zolgensma-treated children in the clinic. Together, these results support that SMA has been particularly amenable to genetic therapy approaches and highlight SMA as a trailblazer for therapeutic development.

Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of presymptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimized Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn-/-;SMN2 or intermediate Smn2B/- SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/- model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart, and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

A study to assess a novel automated electrocardiogram technology in screening for atrial fibrillation.

INTRODUCTION: Atrial fibrillation is often asymptomatic and un-diagnosed in the community resulting in an increased risk of heart failure and stroke to those patients. We evaluated the effectiveness, tolerability, and accuracy of a novel six-channel electrocardiogram digital-health screening device, the RhythmPad, for the detection of atrial fibrillation. METHODS: Seven hundred and fifty-two participants attending the cardiology department were recruited. Two recordings were taken-a six-lead electrocardiogram using the RhythmPad device and a standard 12-lead electrocardiogram. Recorded traces were analyzed by two blinded cardiologists. The computer-generated automated diagnostic reports from both systems were also compared. Post-participation feedback was obtained from study participants using a three-part questionnaire. RESULTS: The sensitivity of the six-lead electrocardiogram compared to the 12-lead electrocardiogram, analyzed by two blinded cardiologists, for the detection of normal sinus rhythm was 95.9%, with a specificity of 97.2%. The sensitivity for the detection of atrial fibrillation using the six-lead ECG was 93.4%, with specificity 96.8%. The six-lead automated diagnostic report had a sensitivity and specificity of 97.5% and 98.6%, respectively, for correctly diagnosing normal sinus rhythm. For the correct diagnosis of atrial fibrillation, the six-lead automated diagnostic report had a sensitivity and specificity of 95.4% and 98.8%, respectively. A total of 95.4% of participants found RhythmPad to be comfortable, with only 0.5% preferring the 12-lead ECG device in comparison to six-lead ECG acquisitions. CONCLUSION: The RhythmPad digital health device and its automated diagnostic report were highly accurate in detecting atrial fibrillation when compared to a standard 12-lead electrocardiogram.

Current Progress in Therapeutic Gene Editing for Monogenic Diseases.

Programmable nucleases allow defined alterations in the genome with ease-of-use, efficiency, and specificity. Their availability has led to accurate and widespread genome engineering, with multiple applications in basic research, biotechnology, and therapy. With regard to human gene therapy, nuclease-based gene editing has facilitated development of a broad range of therapeutic strategies based on both nonhomologous end joining and homology-dependent repair. This review discusses current progress in nuclease-based therapeutic applications for a subset of inherited monogenic diseases including cystic fibrosis, Duchenne muscular dystrophy, diseases of the bone marrow, and hemophilia and highlights associated challenges and future prospects.