Current Clinical Applications of In Vivo Gene Therapy with AAVs.

Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans. For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefit with a single treatment. This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity. There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials. The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic l-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders.

Clinical Follow-Up for Duchenne Muscular Dystrophy Newborn Screening: A Proposal.

New developments in the rapid diagnosis and treatment of boys with Duchenne muscular dystrophy (DMD) have led to growing enthusiasm for instituting DMD newborn screening (NBS) in the United States. Our group has been interested in developing clinical guidance to be implemented consistently in specialty care clinics charged with the care of presymptomatically identified newborns referred after DMD-NBS. We reviewed the existing literature covering patient-centered clinical follow-up after NBS, educational material from public health and advocacy sites, and federal recommendations on effective NBS follow-up. We discussed the review as a group and added our own experience to develop materials suitable for initial parent and primary care provider education. These materials and a series of templates for subspecialist encounters could be used to provide consistent care across centers and serve as the basis for ongoing quality improvement. Muscle Nerve 54: 186-191, 2016.

Neurological Complications of PCR-Proven M. pneumoniae Infections in Children: Prodromal Illness Duration May Reflect Pathogenetic Mechanism.

BACKGROUND: The spectrum of neurologic disease attributable to Mycoplasma pneumoniae in children is incompletely understood in part because of limitations of microbiologic diagnostic methods. Our objective was to characterize the neurologic complications of M. pneumoniae in children using stringent diagnostic criteria. METHODS: All children admitted to the Hospital for Sick Children over a 16-year period with acute neurologic manifestations and polymerase chain reaction (PCR)-confirmed M. pneumoniae infection were eligible for inclusion. Cases were categorized as definite, probable, or possible according to strength of evidence implicating M. pneumoniae. Children with underlying noninfectious neurologic conditions or an alternative infectious cause were excluded. RESULTS: A total of 365 children had M. pneumoniae detected in the cerebrospinal fluid (CSF) or respiratory tract by PCR, 42 (11.5%) of whom had neurologic disease attributable to M. pneumoniae. The most common clinical syndromes were encephalitis (52%), acute disseminated encephalomyelitis (12%), transverse myelitis (12%), and cerebellar ataxia (10%). Two distinct disease patterns were observed, one with a prolonged prodrome (≥7 days), respiratory manifestations, an immunoglobulin M (IgM) response in peripheral blood, and detection of M. pneumoniae in the respiratory tract, but not the CSF, and one with a brief (<7 days) or no prodrome, less frequent respiratory manifestations and IgM response, and detection of M. pneumoniae in the CSF, but not the respiratory tract. CONCLUSIONS: Our findings support the hypothesis of two separate pathogenetic mechanisms for M. pneumoniae-associated neurologic disease, one related to direct infection of the central nervous system and one indirect, likely immunologically mediated.

Milder phenotype of congenital muscular dystrophy in a novel POMT1 mutation.

INTRODUCTION: Congenital muscular dystrophies (CMD) with hypoglycosylated α-dystroglycan due to POMT1 mutations are associated with clinical phenotypes that vary in severity. METHODS: We describe a patient with congenital hypotonia, generalized weakness, elevated creatine kinase (CK), and normal brain imaging. RESULTS: Histochemical analysis of the index case's muscle showed deficiency of glycosylated α-dystroglycan and secondary merosin deficiency. Genetic testing revealed a novel mutation in exon 20 of the POMT1 gene. CONCLUSIONS: Our patient's milder form of CMD adds to the emerging evidence of an expanding phenotype caused by POMT1 mutations. The histopathological findings of the muscle biopsy in this case support the need for careful clinical, genetic, and histochemical diagnostic interpretation.

Five-Year Extension Results of the Phase 1 START Trial of Onasemnogene Abeparvovec in Spinal Muscular Atrophy.

Importance: This ongoing study assesses long-term safety and durability of response in infants with spinal muscular atrophy (SMA) type 1 after dosing with onasemnogene abeparvovec gene replacement therapy. Objective: The primary objective of this ongoing study is to assess safety. The secondary objective is to determine whether developmental milestones achieved in the START phase 1 clinical trial were maintained and new milestones gained. Design, Setting, and Participants: This study is an ongoing, observational, follow-up study for continuous safety monitoring for 15 years in patients from the START phase I study (conducted May 5, 2014, through December 15, 2017) at Nationwide Children's Hospital in Columbus, Ohio. Participants were symptomatic infants with SMA type 1 and 2 copies of SMN2 previously treated with an intravenous dose of onasemnogene abeparvovec (low dose, 6.7 × 1013 vg/kg; or therapeutic dose, 1.1 × 1014 vg/kg) in START. Thirteen of 15 original START patients are included in this analysis; 2 patients' families declined follow-up participation. Data were analyzed from September 21, 2017, to June 11, 2020. Exposures: Median time since dosing of 5.2 (range, 4.6-6.2) years; 5.9 (range, 5.8-6.2) years in the low-dose cohort and 4.8 (range, 4.6-5.6) years in the therapeutic-dose cohort. Main Outcomes and Measures: The primary outcome measure was the incidence of serious adverse events (SAEs). Results: At data cutoff on June 11, 2020, 13 patients treated in START were enrolled in this study (median age, 38.9 [range, 25.4-48.0] months; 7 females; low-dose cohort, n = 3; and therapeutic-dose cohort, n = 10). Serious adverse events occurred in 8 patients (62%), none of which resulted in study discontinuation or death. The most frequently reported SAEs were acute respiratory failure (n = 4 [31%]), pneumonia (n = 4 [31%]), dehydration (n = 3 [23%]), respiratory distress (n = 2 [15%]), and bronchiolitis (n = 2 [15%]). All 10 patients in the therapeutic-dose cohort remained alive and without the need for permanent ventilation. Prior to baseline, 4 patients (40%) in the therapeutic-dose cohort required noninvasive ventilatory support, and 6 patients (60%) did not require regular ventilatory support, which did not change in long-term follow-up. All 10 patients treated with the therapeutic dose maintained previously acquired motor milestones. Two patients attained the new milestone of "standing with assistance" without the use of nusinersen. Conclusions and Relevance: The findings of this ongoing clinical follow-up of patients with SMA type 1 treated with onasemnogene abeparvovec supports the long-term favorable safety profile up to 6 years of age and provides evidence for sustained clinical durability of the therapeutic dose. Trial Registration: ClinicalTrials.gov Identifier: NCT03421977.

Duchenne and Becker muscular dystrophy carriers: Evidence of cardiomyopathy by exercise and cardiac MRI testing.

BACKGROUND: Varied detection methods have resulted in conflicting reports on the prevalence of cardiac disease in Duchenne and Becker muscular dystrophy carriers (MDC). METHODS: We performed a prospective cohort study of 77 genetically-confirmed MDC mothers, 22 non-carrier mothers, and 25 controls. All participants underwent Cardiopulmonary Exercise Testing (CPET) and Cardiac Magnetic Resonance imaging (CMR). RESULTS: 25% of carriers had ventricular ectopy in recovery of exercise (RecVE) as compared to 1 non-carrier and no controls (p = .003). No difference in age or maximal oxygen consumption was noted. 11 carriers had abnormal (<55%) left ventricular ejection fraction by CMR. Evidence of late gadolinium enhancement (LGE) was noted in 48% of MDC, 1 non-carrier patient and no control subjects (p < .0001). Subset analysis of LGE+ and LGE- subjects revealed differences in age (44.1 v 38.6 yrs.; p = .005), presence of RecVE, (38.9% v 10.5%, p = .004), and high serum creatine kinase (CK) (> 289 U/l; 52.8% v 31.6%, p = .065). CONCLUSION: We describe the prevalence of disease using CPET and CMR in genetically-proven MDC. 49% of carriers had fibrosis, opposed to 5% of non-carriers, highlighting the importance of genetic testing in this population. Despite cardiomyopathy, functional assessment by treadmill was normal, illustrating the discrepancy in cardiac and skeletal muscle impacts. Age, RecVE and serum CK appear to have an important role in predicting cardiomyopathy. Serum CK levels suggest that a systemic higher global disease severity and not tissue heterogeneity may be the etiology for greater cardiac disease and relatively spared skeletal muscle disease in this population. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT02972580?term=mendell&cond=Duchenne+Muscular+Dystrophy&rank=5; ClinicalTrials.gov Identifier: NCT02972580.

From Clinical Trials to Clinical Practice: Practical Considerations for Gene Replacement Therapy in SMA Type 1.

Spinal muscular atrophy is a devastating neurodegenerative autosomal recessive disease that results from survival of motor neuron 1 (SMN1) gene mutation or deletion. Patients with spinal muscular atrophy type 1 utilizing supportive care, which focuses on symptom management, never sit unassisted, and 75% die or require permanent ventilation by age 13.6 months. Onasemnogene abeparvovec (Zolgensma, formerly AVXS-101) is a gene replacement therapy comprising an adeno-associated viral vector containing the human SMN gene under control of the chicken beta-actin promoter. This therapy addresses the genetic root cause of the disease by increasing functional SMN protein in motor neurons and preventing neuronal cell death, resulting in improved neuronal and muscular function as previously demonstrated in transgenic animal models. In an open-label, one-arm, dose-escalation phase 1 trial, systemic administration of onasemnogene abeparvovec via a one-time infusion over one hour demonstrated improved motor function and survival in all infants symptomatic for spinal muscular atrophy type 1. Of the 12 patients who received the proposed therapeutic dose, 11 achieved independent sitting, two achieved independent standing, and two are able to walk. Most of these 12 patients remained free of respiratory supportive care. The only treatment-related adverse event observed was transient asymptomatic transaminasemia that resolved with a short course of prednisolone treatment. This review discusses the biological rationale underlying gene replacement therapy for spinal muscular atrophy, describes the onasemnogene abeparvovec clinical trial experience, and provides expert recommendations as a reference for the real-world use of onasemnogene abeparvovec in clinical practice. As of May 24, 2019, the Food and Drug Administration approved onasemnogene abeparvovec, the first gene therapy approved to treat children younger than two years with spinal muscular atrophy.

AVXS-101 (Onasemnogene Abeparvovec) for SMA1: Comparative Study with a Prospective Natural History Cohort.

BACKGROUND: Spinal muscular atrophy type 1 (SMA1) is the leading genetic cause of infant mortality for which therapies, including AVXS-101 (onasemnogene abeparvovec, Zolgensma®) gene replacement therapy, are emerging. OBJECTIVE: This study evaluated the effectiveness of AVXS-101 in infants with spinal muscular atrophy type 1 (SMA1) compared with a prospective natural history cohort and a cohort of healthy infants. METHODS: Twelve SMA1 infants received the proposed therapeutic dose of AVXS-101 (NCT02122952). Where possible, the following outcomes were compared with a natural history cohort of SMA1 infants (n = 16) and healthy infants (n = 27) enrolled in the NeuroNEXT (NN101) study (NCT01736553): event-free survival, CHOP-INTEND scores, motor milestone achievements, compound muscle action potential (CMAP), and adverse events. RESULTS: Baseline characteristics of SMA1 infants in the AVXS-101 and NN101 studies were similar in age and genetic profile. The proportion of AVXS-101-treated infants who survived by 24 months of follow-up was higher compared with the NN101 study (100% vs 38%, respectively). The average baseline CHOP-INTEND score for NN101 SMA1 infants was 20.3, worsening to 5.3 by age 24 months; the average baseline score in AVXS-101-treated infants was 28.2, improving to 56.5 by age 24 months. Infants receiving AVXS-101 achieved motor milestones, such as sitting unassisted and walking. Improvements in CMAP peak area were observed in AVXS-101-treated infants at 6 and 24 months (means of 1.1 and 3.2 mV/s, respectively). CONCLUSIONS: In this study, AVXS-101 increased the probability of survival, rapidly improved motor function, and enabled motor milestone achievement in SMA1 infants.

Gene Delivery for Limb-Girdle Muscular Dystrophy Type 2D by Isolated Limb Infusion.

In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/µg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

A Roadmap to Newborn Screening for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy, with an estimated frequency of 1:5000 live births. The impact of the disease presents as early as infancy with significant developmental delays, and ultimately loss of ambulation and respiratory insufficiency. Glucocorticoids are the only pharmacological agents known to alter the natural progression of the disease by prolonging ambulation, reducing scoliosis, and assisted ventilation. Introduction of therapy at an early age may halt the muscle pathology in DMD. In anticipation of the potentially disease-modifying products that are reaching regulatory review, Parent Project Muscular Dystrophy (PPMD) formally initiated a national Duchenne Newborn Screening (DNBS) effort in December 2014 to build public health infrastructure for newborn screening (NBS) for Duchenne in the United States. The effort includes a formalized national Duchenne Newborn Screening Steering Committee, six related Working Groups, a Duchenne Screening Test Development Project led by PerkinElmer, a program with the American College of Medical Genetic and Genomics' Newborn Screening Translation Research Network (NBSTRN), and collaborations with other Duchenne partners and federal agencies involved in NBS. We herein review the organization and effort of the U.S. DNBS program to develop the evidence supporting the implementation of NBS for DMD.

Follistatin Gene Therapy Improves Ambulation in Becker Muscular Dystrophy.

Follistatin is a ubiquitous secretory propeptide that functions as a potent inhibitor of the myostatin pathway, resulting in an increase in skeletal muscle mass. Its ability to interact with the pituitary activin-inhibin axis and suppress the secretion of follicle-stimulating hormone (FSH) called for caution in its clinical applicability. This limitation was circumvented by the use of one of the alternatively spliced follistatin variants, FS344, undergoing post-translational modification to FS315. This follistatin isoform is serum-based, and has a 10-fold lower affinity to activin compared to FS288. Preclinical studies of intramuscular delivery of the follistatin gene demonstrated safety and efficacy in enhancing muscle mass. We herein review the evidence supporting the utility of follistatin as a genetic enhancer to improve cellular performance. In addition, we shed light on the results of the first clinical gene transfer trial using the FS344 isoform of follistatin in subjects with Becker muscular dystrophy as well as the future directions for clinical gene therapy trials using follistatin.

A slowly progressive form of limb-girdle muscular dystrophy type 2C associated with founder mutation in the SGCG gene in Puerto Rican Hispanics.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is considered one of the severe forms of childhood-onset muscular dystrophy. The geographical distribution of founder mutations in the SGCG gene has a prominent effect on the prevalence of LGMD2C in certain populations. The aim of this study was to confirm the hypothesis that the c.787G>A (p.E263K) mutation in the SGCG gene is a founder mutation among Puerto Rican Hispanics and to characterize the associated clinical and immunohistochemical phenotype. Genotyping of six polymorphic microsatellite markers internal to (D13S232) and flanking (D13S175, D13S292, D13S787, D13S1243, D13S283) the SGCG gene was performed on four unrelated Puerto Rican patients with LGMD2C. Preserved ambulation to the second decade of life was observed in at least two subjects. Immunostaining of skeletal muscle demonstrated absence of γ-sarcoglycan in all affected subjects. Two markers, D13S232 and D13S292, were highly informative and confirmed that all four families share the haplotype of the mutant allele. Our findings confirm that the E263K missense mutation in the SGCG gene is a founder mutation in Puerto Rican Hispanics. A slowly progressive disease course with prolonged preservation of ambulation can be seen in association with this mutation, providing evidence for phenotypic variability.