Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types.

With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disease caused by loss of the enzyme aspartylglucosaminidase (AGA), resulting in AGA substrate accumulation. AGU patients have a slow but progressive neurodegenerative disease course, for which there is no approved disease-modifying treatment. In this study, AAV9/AGA was administered to Aga-/- mice intravenously (i.v.) or intrathecally (i.t.), at a range of doses, either before or after disease pathology begins. At either treatment age, AAV9/AGA administration led to (1) dose dependently increased and sustained AGA activity in body fluids and tissues; (2) rapid, sustained, and dose-dependent elimination of AGA substrate in body fluids; (3) significantly rescued locomotor activity; (4) dose-dependent preservation of Purkinje neurons in the cerebellum; and (5) significantly reduced gliosis in the brain. Treated mice had no abnormal neurological phenotype and maintained body weight throughout the whole experiment to 18 months old. In summary, these results demonstrate that treatment of Aga-/- mice with AAV9/AGA is effective and safe, providing strong evidence that AAV9/AGA gene therapy should be considered for human translation. Further, we provide a direct comparison of the efficacy of an i.v. versus i.t. approach using AAV9, which should greatly inform the development of similar treatments for other related lysosomal storage diseases.

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.

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.

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.

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and β subunits of the β-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the "correction efficacy" of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.

Gene therapy clinical trials for neurological disorders are ongoing using intrathecal injection of adeno-associated virus (AAV) vector directly into the cerebral spinal fluid. Preliminary findings from these trials and results from extensive animal studies provides compelling data supporting the safety and benefit of intrathecal delivery of AAV vectors for inherited neurological disorders. Intrathecal delivery can be achieved by a lumbar puncture (LP) or intracisterna magna (ICM) injection, although ICM is not commonly used in clinical practice due to increased procedural risk. Few studies directly compared these delivery methods and there are limited reports on transduction of the PNS. To further test the utility of ICM or LP delivery for neuropathies, we performed a head to head comparison of AAV serotype 9 (AAV9) vectors expressing GFP injected into the cisterna magna or lumbar subarachnoid space in mice. We report that an intrathecal gene delivery of AAV9 in mice leads to stable transduction of neurons and glia in the brain and spinal cord and has a widespread distribution that includes components of the PNS. Vector expression was notably higher in select brain and PNS regions following ICM injection, while higher amounts of vector was found in the lower spinal cord and peripheral organs following LP injection. These findings support that intrathecal AAV9 delivery is a translationally relevant delivery method for inherited neuropathies.

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.

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.

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).

This brief review of current research progress on Charcot-Marie-Tooth (CMT) disease is a summary of discussions initiated at the Hereditary Neuropathy Foundation (HNF) scientific advisory board meeting on November 7, 2014. It covers recent published and unpublished in vitro and in vivo research. We discuss recent promising preclinical work for CMT1A, the development of new biomarkers, the characterization of different animal models, and the analysis of the frequency of gene mutations in patients with CMT. We also describe how progress in related fields may benefit CMT therapeutic development, including the potential of gene therapy and stem cell research. We also discuss the potential to assess and improve the quality of life of CMT patients. This summary of CMT research identifies some of the gaps which may have an impact on upcoming clinical trials. We provide some priorities for CMT research and areas which HNF can support. The goal of this review is to inform the scientific community about ongoing research and to avoid unnecessary overlap, while also highlighting areas ripe for further investigation. The general collaborative approach we have taken may be useful for other rare neurological diseases.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU. We have here characterized the consequences of a novel AGU mutation that results in Thr122Lys exchange in AGA, and compared this mutant form to one carrying the worldwide most common AGU mutation, AGU-Fin. We show that T122K mutated AGA is expressed in normal amounts and localized in lysosomes, but exhibits low AGA activity due to impaired processing of the precursor molecule into subunits. Coexpression of T122K with wildtype AGA results in processing of the precursor into subunits, implicating that the mutation causes a local misfolding that prevents the precursor from becoming processed. Similar data were obtained for the AGU-Fin mutant polypeptide. We have here also identified small chemical compounds that function as chemical or pharmacological chaperones for the mutant AGA. Treatment of patient fibroblasts with these compounds results in increased AGA activity and processing, implicating that these substances may be suitable for chaperone mediated therapy for AGU.

Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200-800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.Molecular Therapy - Nucleic Acids (2013) 2, e70; doi:10.1038/mtna.2012.61; published online 5 February 2013.

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood-brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1-6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid-induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)-packaged clones were administered directly into brain or i.v. 1 day after kainic acid-induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells.