Intrathecal Gene Therapy for Giant Axonal Neuropathy.

BACKGROUND: Giant axonal neuropathy is a rare, autosomal recessive, pediatric, polysymptomatic, neurodegenerative disorder caused by biallelic loss-of-function variants in GAN, the gene encoding gigaxonin. METHODS: We conducted an intrathecal dose-escalation study of scAAV9/JeT-GAN (a self-complementary adeno-associated virus-based gene therapy containing the GAN transgene) in children with giant axonal neuropathy. Safety was the primary end point. The key secondary clinical end point was at least a 95% posterior probability of slowing the rate of change (i.e., slope) in the 32-item Motor Function Measure total percent score at 1 year after treatment, as compared with the pretreatment slope. RESULTS: One of four intrathecal doses of scAAV9/JeT-GAN was administered to 14 participants - 3.5×1013 total vector genomes (vg) (in 2 participants), 1.2×1014 vg (in 4), 1.8×1014 vg (in 5), and 3.5×1014 vg (in 3). During a median observation period of 68.7 months (range, 8.6 to 90.5), of 48 serious adverse events that had occurred, 1 (fever) was possibly related to treatment; 129 of 682 adverse events were possibly related to treatment. The mean pretreatment slope in the total cohort was -7.17 percentage points per year (95% credible interval, -8.36 to -5.97). At 1 year after treatment, posterior mean changes in slope were -0.54 percentage points (95% credible interval, -7.48 to 6.28) with the 3.5×1013-vg dose, 3.23 percentage points (95% credible interval, -1.27 to 7.65) with the 1.2×1014-vg dose, 5.32 percentage points (95% credible interval, 1.07 to 9.57) with the 1.8×1014-vg dose, and 3.43 percentage points (95% credible interval, -1.89 to 8.82) with the 3.5×1014-vg dose. The corresponding posterior probabilities for slowing the slope were 44% (95% credible interval, 43 to 44); 92% (95% credible interval, 92 to 93); 99% (95% credible interval, 99 to 99), which was above the efficacy threshold; and 90% (95% credible interval, 89 to 90). Between 6 and 24 months after gene transfer, sensory-nerve action potential amplitudes increased, stopped declining, or became recordable after being absent in 6 participants but remained absent in 8. CONCLUSIONS: Intrathecal gene transfer with scAAV9/JeT-GAN for giant axonal neuropathy was associated with adverse events and resulted in a possible benefit in motor function scores and other measures at some vector doses over a year. Further studies are warranted to determine the safety and efficacy of intrathecal AAV-mediated gene therapy in this disorder. (Funded by the National Institute of Neurological Disorders and Stroke and others; ClinicalTrials.gov number, NCT02362438.).

Intrathecal delivery of a bicistronic AAV9 vector expressing ß-hexosaminidase A corrects Sandhoff disease in a murine model: A dosage study.

The pathological accumulation of GM2 ganglioside associated with Tay-Sachs disease (TSD) and Sandhoff disease (SD) occurs in individuals who possess mutant forms of the heterodimer ß-hexosaminidase A (Hex A) because of mutation of the HEXA and HEXB genes, respectively. With a lack of approved therapies, patients experience rapid neurological decline resulting in early death. A novel bicistronic vector carrying both HEXA and HEXB previously demonstrated promising results in mouse models of SD following neonatal intravenous administration, including significant reduction in GM2 accumulation, increased levels of Hex A, and a 2-fold extension of survival. The aim of the present study was to identify an optimal dose of the bicistronic vector in 6-week-old SD mice by an intrathecal route of administration along with transient immunosuppression, to inform possible clinical translation. Three doses of the bicistronic vector were tested: 2.5e11, 1.25e11, and 0.625e11 vector genomes per mouse. The highest dose provided the greatest increase in biochemical and behavioral parameters, such that treated mice lived to a median age of 56 weeks (>3 times the lifespan of the SD controls). These results have direct implications in deciding a human equivalent dose for TSD/SD and have informed the approval of a clinical trial application (NCT04798235).

CSTB gene replacement improves neuroinflammation, neurodegeneration and ataxia in murine type 1 progressive myoclonus epilepsy.

EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease.

Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease.

Mutations in the CLN5 gene cause the fatal, pediatric, neurodegenerative disease CLN5 neuronal ceroid lipofuscinosis. Affected children suffer progressive neuronal loss, visual failure and premature death. Presently there is no treatment. This study evaluated dual intracerebroventricular (ICV) and intravitreal (IVT) administration of a self-complementary adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) into CLN5 affected sheep (CLN5-/-) at various disease stages. CLN5 disease progression was slowed in pre-symptomatic sheep who received a moderate dose of scAAV9/oCLN5, whilst a higher ICV dose treatment in early and advanced symptomatic animals delayed or halted disease progression. Intracranial (brain) volume loss was attenuated in all treatment cohorts, and visual function was also sustained in both the early and advanced symptomatic treated sheep over the 24-month duration of the study. Robust CLN5 protein expression was detected throughout the brain and spinal cord, and improvements in central nervous system and retinal disease correlates were observed. These findings hold translational promise for extending and improving the quality of life in both pre-symptomatic and symptomatic CLN5 patients, and prompted the initiation of the first in-human Phase I/II clinical trial testing ICV/IVT administration of scAAV9 encoding human CLN5 (https://clinicaltrials.gov/; NCT05228145).

Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis.

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-HEXA, HEXB, and GM2A-within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 1014 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a-/-). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a-/- mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.

AAV-based in vivo gene therapy for neurological disorders.

Recent advancements in gene supplementation therapy are expanding the options for the treatment of neurological disorders. Among the available delivery vehicles, adeno-associated virus (AAV) is often the favoured vector. However, the results have been variable, with some trials dramatically altering the course of disease whereas others have shown negligible efficacy or even unforeseen toxicity. Unlike traditional drug development with small molecules, therapeutic profiles of AAV gene therapies are dependent on both the AAV capsid and the therapeutic transgene. In this rapidly evolving field, numerous clinical trials of gene supplementation for neurological disorders are ongoing. Knowledge is growing about factors that impact the translation of preclinical studies to humans, including the administration route, timing of treatment, immune responses and limitations of available model systems. The field is also developing potential solutions to mitigate adverse effects, including AAV capsid engineering and designs to regulate transgene expression. At the same time, preclinical research is addressing new frontiers of gene supplementation for neurological disorders, with a focus on mitochondrial and neurodevelopmental disorders. In this Review, we describe the current state of AAV-mediated neurological gene supplementation therapy, including critical factors for optimizing the safety and efficacy of treatments, as well as unmet needs in this field.

SMRT Sequencing Enables High-Throughput Identification of Novel AAVs from Capsid Shuffling and Directed Evolution.

The use of AAV capsid libraries coupled with various selection strategies has proven to be a remarkable approach for generating novel AAVs with enhanced and desired features. The inability to reliably sequence the complete capsid gene in a high-throughput manner has been the bottleneck of capsid engineering. As a result, many library strategies are confined to localized and modest alterations in the capsid, such as peptide insertions or single variable region (VR) alterations. The caveat of short reads by means of next-generation sequencing (NGS) hinders the diversity of capsid library construction, shifting the field away from whole-capsid modifications. We generated AAV capsid shuffled libraries of naturally occurring AAVs and applied directed evolution in both mice and non-human primates (NHPs), with the goal of yielding AAVs that are compatible across both species for translational applications. We recovered DNA from the tissues of injected animal and used single molecule real-time (SMRT) sequencing to identify variants enriched in the central nervous system (CNS). We provide insights and considerations for variant identification by comparing bulk tissue sequencing to that of isolated nuclei. Our work highlights the potential advantages of whole-capsid engineering, as well as indispensable methodological improvements for the analysis of recovered capsids, including the nuclei-enrichment step and SMRT sequencing.

Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease.

CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is characterised by progressive neuronal loss, blindness, and premature death. There is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected (CLN5-/-) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three disease stages. The treatment delayed disease progression, extended survival and slowed stereotypical brain atrophy in most animals. Of note, one high dose treated animal only developed mild disease symptomology and survived to 60.1 months of age, triple the natural life expectancy of an untreated CLN5-/- sheep. Eyesight was not preserved at any administration age or dosage. Histopathologic examination revealed that greater transduction efficiency was achieved through higher ICV doses, and this resulted in greater amelioration of disease pathology. Together with other pre-clinical data from CLN5-/- sheep, the safety and efficacy data from these investigational new drug (IND)-enabling studies supported the initiation of the first in-human CLN5 gene therapy clinical study using the ICV delivery route for the treatment of CLN5 NCL. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05228145.

Biochemical Correction of GM2 Ganglioside Accumulation in AB-Variant GM2 Gangliosidosis.

GM2 gangliosidosis is a group of genetic disorders that result in the accumulation of GM2 ganglioside (GM2) in brain cells, leading to progressive central nervous system (CNS) atrophy and premature death in patients. AB-variant GM2 gangliosidosis (ABGM2) arises from loss-of-function mutations in the GM2 activator protein (GM2AP), which is essential for the breakdown of GM2 in a key catabolic pathway required for CNS lipid homeostasis. In this study, we show that intrathecal delivery of self-complementary adeno-associated virus serotype-9 (scAAV9) harbouring a functional human GM2A transgene (scAAV9.hGM2A) can prevent GM2 accumulation in in GM2AP-deficient mice (Gm2a-/- mice). Additionally, scAAV9.hGM2A efficiently distributes to all tested regions of the CNS within 14 weeks post-injection and remains detectable for the lifespan of these animals (up to 104 weeks). Remarkably, GM2AP expression from the transgene scales with increasing doses of scAAV9.hGM2A (0.5, 1.0 and 2.0 × 1011 vector genomes (vg) per mouse), and this correlates with dose-dependent correction of GM2 accumulation in the brain. No severe adverse events were observed, and comorbidities in treated mice were comparable to those in disease-free cohorts. Lastly, all doses yielded corrective outcomes. These data indicate that scAAV9.hGM2A treatment is relatively non-toxic and tolerable, and biochemically corrects GM2 accumulation in the CNS-the main cause of morbidity and mortality in patients with ABGM2. Importantly, these results constitute proof-of-principle for treating ABGM2 with scAAV9.hGM2A by means of a single intrathecal administration and establish a foundation for future preclinical research.

CNS-dominant human FMRP isoform rescues seizures, fear, and sleep abnormalities in Fmr1-KO mice.

Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.

Intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 shows safety and efficacy in preclinical studies.

Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay, and subsequent intellectual disability, secondary microcephaly, and epilepsy. We preformed preclinical studies evaluating an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50 and describe in vitro studies that demonstrate transduction of patient-derived fibroblasts with AAV2/AP4M1, resulting in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1-KO mice were intrathecally (i.t.) injected with 5 × 1011, 2.5 × 1011, or 1.25 × 1011 vector genome (vg) doses of AAV9/AP4M1 at P7-P10 or P90. Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in WT mice, rats, and nonhuman primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1 × 1015 vg. Of note, similar degrees of minimal-to-mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future i.t. AAV studies. These preclinical results identify an acceptably safe and efficacious dose of i.t.-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.

Biodistribution of Adeno-Associated Virus Gene Therapy Following Cerebrospinal Fluid-Directed Administration.

Adeno-associated virus (AAV)-based gene therapies, exemplified by the approved therapy for spinal muscular atrophy, have the potential to deliver disease-course-altering treatments for central nervous system (CNS) indications. However, several clinical trials have reported severe adverse events, including patient deaths following high-dose systemic administration for muscle-directed gene transfer, highlighting the need to explore approaches utilizing lower doses when targeting the CNS. Animal models of disease provide insight into the response to new AAV therapies. However, translation from small to larger animals and eventually to humans is hampered by anatomical and biological differences across the species and their impact on AAV delivery. We performed a literature review of preclinical studies of AAV gene therapy biodistribution following cerebrospinal fluid (CSF) delivery (intracerebroventricular, intra-cisterna magna, and intrathecal lumbar). The reviewed literature varies greatly in the reported biodistribution of AAV following administration into the CSF. Differences between studies, including animal model, vector serotype used, method used to assess biodistribution, and route of administration, among other variables, contribute to differing outcomes and difficulties in translating these preclinical results. For example, only half of the published AAV-based gene therapy studies report vector copy number, the most direct readout following administration of a vector; none of these studies reported details such as the empty:full capsid ratio and quality of encapsidated genome. Analysis of the last decade's literature focusing on AAV-based gene therapies targeting the CNS underscores limitations of the body of knowledge and room for continued research. In particular, there is a need to understand the biodistribution achieved by different CSF-directed routes of administration and determining if specific cell types/structures of interest will be transduced. Our findings point to a clear need for a more systematic approach across the field to align the assessments and elements reported in preclinical research to enable more reliable translation across animal models and into human studies.

The Efficacy of a Human-Ready miniMECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome.

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Long-term progression of retinal degeneration in a preclinical model of CLN7 Batten disease as a baseline for testing clinical therapeutics.

BACKGROUND: Batten disease is characterized by cognitive and motor impairment, retinal degeneration, and seizures leading to premature death. Recent studies have shown efficacy for a gene therapy approach for CLN7 Batten disease. This gene therapy approach is promising to treat cognitive and motor impairment, but is not likely to delay vision loss. Additionally, the natural progression of retinal degeneration in CLN7 Batten disease patients is not well-known. METHODS: We performed visual examinations on five patients with CLN7 Batten disease and found that patients were far progressed in degeneration within their first five years of life. To better understand the disease progression, we characterized the retina of a preclinical mouse model of CLN7 Batten disease, through the age at which mice present with paralysis and premature death. FINDINGS: We found that this preclinical model shows signs of photoreceptor to bipolar synaptic defects early, and displays rod-cone dystrophy with late loss of bipolar cells. This vision loss could be followed not only via histology, but using clinical live imaging similar to that used in human patients. INTERPRETATION: Natural history studies of rare paediatric neurodegenerative conditions are complicated by the rapid degeneration and limited availability of patients. Characterization of degeneration in the preclinical model allows for future experiments to better understand the mechanisms underlying the retinal disease progression in order to find therapeutics to treat patients, as well as to evaluate these therapeutic options for future human clinical trials. FUNDING: Van Sickle Family Foundation Inc., NIHP30EY030413, Morton Fichtenbaum Charitable Trust and 5T32GM131945-03.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders.

We are in an emerging era of gene-based therapeutics with significant promise for rare genetic disorders. The potential is particularly significant for genetic central nervous system disorders that have begun to achieve Food and Drug Administration approval for select patient populations. This review summarizes the discussions and presentations of the National Institute of Mental Health-sponsored workshop "Gene-Based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders," which was held in January 2021. Here, we distill the points raised regarding various precision medicine approaches related to neurodevelopmental and psychiatric disorders that may be amenable to gene-based therapies.

AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease.

Neuronal ceroid lipofuscinosis type 7 (CLN7) disease is a lysosomal storage disease caused by mutations in the facilitator superfamily domain containing 8 (MFSD8) gene, which encodes a membrane-bound lysosomal protein, MFSD8. To test the effectiveness and safety of adeno-associated viral (AAV) gene therapy, an in vitro study demonstrated that AAV2/MFSD8 dose dependently rescued lysosomal function in fibroblasts from a CLN7 patient. An in vivo efficacy study using intrathecal administration of AAV9/MFSD8 to Mfsd8- /- mice at P7-P10 or P120 with high or low dose led to clear age- and dose-dependent effects. A high dose of AAV9/MFSD8 at P7-P10 resulted in widespread MFSD8 mRNA expression, tendency of amelioration of subunit c of mitochondrial ATP synthase accumulation and glial fibrillary acidic protein immunoreactivity, normalization of impaired behaviors, doubled median life span, and extended normal body weight gain. In vivo safety studies in rodents concluded that intrathecal administration of AAV9/MFSD8 was safe and well tolerated. In summary, these results demonstrated that the AAV9/MFSD8 vector is both effective and safe in preclinical models.

Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice Using an Intravenous Self-Complementary Hexosaminidase Vector.

BACKGROUND: GM2 gangliosidosis is a neurodegenerative, lysosomal storage disease caused by the deficiency of ß-hexosaminidase A enzyme (Hex A), an α/ß-subunit heterodimer. A novel variant of the human hexosaminidase α-subunit, coded by HEX M, has previously been shown to form a stable homodimer, Hex M, that hydrolyzes GM2 gangliosides (GM2) in vivo. MATERIALS & METHODS: The current study assessed the efficacy of intravenous (IV) delivery of a self-complementary adeno-associated virus serotype 9 (scAAV9) vector incorporating the HEXM transgene, scAAV9/HEXM, including the outcomes based on the dosages provided to the Sandhoff (SD) mice. Six-week-old SD mice were injected with either 2.5E+12 vector genomes (low dose, LD) or 1.0E+13 vg (high dose, HD). We hypothesized that when examining the dosage comparison for scAAV9/HEXM in adult SD mice, the HD group would have more beneficial outcomes than the LD cohort. Assessments included survival, behavioral outcomes, vector biodistribution, and enzyme activity within the central nervous system. RESULTS: Toxicity was observed in the HD cohort, with 8 of 14 mice dying within one month of the injection. As compared to untreated SD mice, which have typical survival of 16 weeks, the LD cohort and the remaining HD mice had a significant survival benefit with an average/median survival of 40.6/34.5 and 55.9/56.7 weeks, respectively. Significant behavioral, biochemical and molecular benefits were also observed. The second aim of the study was to investigate the effects of IV mannitol infusions on the biodistribution of the LD scAAV9/HEXM vector and the survival of the SD mice. Increases in both the biodistribution of the vector as well as the survival benefit (average/median of 41.6/49.3 weeks) were observed. CONCLUSION: These results demonstrate the potential benefit and critical limitations of the treatment of GM2 gangliosidosis using IV delivered AAV vectors.

Comparative outcome analysis of osseointegrated reconstruction and replantation for digital amputations.

The long-term outcomes of osseointegration for digital amputations are not well established, and it is not known whether osseointegration can achieve similar function and patient satisfaction to conventional surgical options such as replantation and microsurgical toe transfer. We compared the long-term outcomes after digital osseointegration and replantation. Six patients treated by osseointegration and seven patients treated by replantation were included, with median follow-ups of 8 years and 4.6 years, respectively. Outcomes were assessed using the Michigan Hand Outcomes Questionnaire, grip and pinch strength, range of motion, two-point discrimination, Semmes-Weinstein tests, Jebsen-Taylor Hand Function Test and clinical photography. Osseointegration was associated with poorer sensibility and range of motion than replantation; no other differences reached statistical significance. Long-term osseointegration is a safe and effective reconstructive option that can deliver excellent outcomes in appropriately selected patients.Level of evidence: IV.

Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice.

Loss of the maternal UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.

Adeno-associated viral vector serotype 9-based gene replacement therapy for SURF1-related Leigh syndrome.

SURF1 (surfeit locus protein 1)-related Leigh syndrome is an early-onset neurodegenerative disorder, characterized by reduction in complex IV activity, resulting in disrupted mitochondrial function. Currently, there are no treatment options available. To test our hypothesis that adeno-associated viral vector serotype 9 (AAV9)/human SURF1 (hSURF1) gene replacement therapy can provide a potentially meaningful and long-term therapeutic benefit, we conducted preclinical efficacy studies using SURF1 knockout mice and safety evaluations with wild-type (WT) mice. Our data indicate that with a single intrathecal (i.t.) administration, our treatment partially and significantly rescued complex IV activity in all tissues tested, including liver, brain, and muscle. Accordingly, complex IV content (examined via MT-CO1 protein expression level) also increased with our treatment. In a separate group of mice, AAV9/hSURF1 mitigated the blood lactic acidosis induced by exhaustive exercise at 9 months post-dosing. A toxicity study in WT mice showed no adverse effects in either the in-life portion or after microscopic examination of major tissues up to a year following the same treatment regimen. Taken together, our data suggest a single dose, i.t. administration of AAV9/hSURF1 is safe and effective in improving biochemical abnormalities induced by SURF1 deficiency with potential applicability for SURF1-related Leigh syndrome patients.