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BACKGROUND: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8(−/y)) male mice increased triiodothyronine (T(3)) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. METHODS: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8(−/y)/Organic anion-transporting polypeptide 1c1 (Oatp1c1)(−/ −) double knockout (dKO) mouse model of AHDS, which, unlike Mct8(−/y) mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120–P140 after which tissues were collected and analyzed. RESULTS: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120–P140, which was accompanied by a near normalization of T(3) content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T(3)/reverse T(3) (rT(3)) ratio and in liver deiodinase 1 expression. CONCLUSION: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Spinal Muscular Atrophy (SMA) is the leading genetic cause of infant mortality. The most common form of SMA is caused by mutations in the SMN1 gene, located on 5q (SMA). On the other hand, mutations in IGHMBP2 lead to a large disease spectrum with no clear genotype–phenotype correlation, which includes Spinal Muscular Atrophy with Muscular Distress type 1 (SMARD1), an extremely rare form of SMA, and Charcot–Marie–Tooth 2S (CMT2S). We optimized a patient-derived in vitro model system that allows us to expand research on disease pathogenesis and gene function, as well as test the response to the AAV gene therapies we have translated to the clinic. We generated and characterized induced neurons (iN) from SMA and SMARD1/CMT2S patient cell lines. After establishing the lines, we treated the generated neurons with AAV9-mediated gene therapy (AAV9.SMN (Zolgensma) for SMA and AAV9.IGHMBP2 for IGHMBP2 disorders (NCT05152823)) to evaluate the response to treatment. The iNs of both diseases show a characteristic short neurite length and defects in neuronal conversion, which have been reported in the literature before with iPSC modeling. SMA iNs respond to treatment with AAV9.SMN in vitro, showing a partial rescue of the morphology phenotype. For SMARD1/CMT2S iNs, we were able to observe an improvement in the neurite length of neurons after the restoration of IGHMBP2 in all disease cell lines, albeit to a variable extent, with some lines showing better responses to treatment than others. Moreover, this protocol allowed us to classify a variant of uncertain significance on IGHMBP2 on a suspected SMARD1/CMT2S patient. This study will further the understanding of SMA, and SMARD1/CMT2S disease in particular, in the context of variable patient mutations, and might further the development of new treatments, which are urgently needed.

Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55–75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

Allan-Herndon-Dudley syndrome (AHDS) is a severe X-linked intellectual and psychomotor disability disorder accompanied by abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in the monocarboxylate transporter 8 (MCT8), a specific TH transporter widely expressed in the central nervous system. MCT8 gene mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. AAV9-based gene therapy is a promising approach to treat monogenic neurological disorders. Here, the potential of this approach was tested in the well-established double knockout (dKO) Mct8-/y;Oatp1c1-/- mouse model of AHDS, which displays disease-relevant neurological and TH phenotypes. Systemic intravenous delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive function, as well as rescue of T3-brain content and T3-related gene expression. This preclinical study indicates that this gene therapy may improve the neurological symptoms of AHDS patients.

CLN6-Batten disease, a form of neuronal ceroid lipofuscinosis is a rare lysosomal storage disorder presenting with gradual declines in motor, visual, and cognitive abilities and early death by 12–15 years of age. We developed a self-complementary adeno-associated virus serotype 9 (scAAV9) vector expressing the human CLN6 gene under the control of a chicken β-actin (CB) hybrid promoter. Intrathecal delivery of scAAV9.CB.hCLN6 into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 4-year-old non-human primates was safe, well tolerated, and led to efficient targeting throughout the brain and spinal cord. A single intracerebroventricular (i.c.v.) injection at post-natal day 1 in Cln6 mutant mice delivered scAAV9.CB.CLN6 directly into the CSF, and it prevented or drastically reduced all of the pathological hallmarks of Batten disease. Moreover, there were significant improvements in motor performance, learning and memory deficits, and survival in treated Cln6 mutant mice, extending survival from 15 months of age (untreated) to beyond 21 months of age (treated). Additionally, many parameters were similar to wild-type counterparts throughout the lifespan of the treated mice., Graphical Abstract, CLN6-Batten disease is a fatal lysosomal storage disorder that primarily affects children. Weimer and colleagues developed an AAV9 gene therapy that was well tolerated in mice and non-human primates. Delivery of this therapy into the brain of CLN6-Batten mice prevented most neurodegenerative symptoms, with treated mice living a full lifespan.

In May of 2019, the adeno-associated virus (AAV)-based gene therapy onasemnogene abeparvovec-xioi (Zolgensma) became the second Food and Drug Administration (FDA)-approved gene therapy with designated use for infants diagnosed with spinal muscular atrophy (SMA). The decision came nearly 10 years after results of the first preclinical models were initially reported. While the journey was an arduous one, the approval was an indication of the remarkable success of the first in-human clinical trials. According to the traditional classification system of autosomal recessive SMA, of which there are multiple types with phenotypic variability, SMA type 1 is the most common and most severe and represents 45% of the SMA patient population. Children with SMA type 1 cannot lift their heads without assistance and do not live past their second birthday. With Zolgensma, the first treated children with SMA type 1 have reached 5 years of age and some of them achieved the ability to sit unassisted or even walk. In this article, we review the work that led to FDA approval with emphasis on the development of preclinical and clinical studies.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

NEDAMSS is a newly discovered neurodevelopmental disorder with regression, abnormal movements, loss of speech and seizures. The disease is caused by heterozygous mutations in the Interferon Regulatory Factor 2 Binding Protein-Like (IRF2BPL) gene. IRF2BPL is a ubiquitously expressed intron-less transcriptional regulator. The function of the protein is currently unknown, but the observed disease phenotype indicates a role in development and maintenance of the nervous system. To study this disease, we used patient skin-derived fibroblasts and reprogrammed them into induced neuronal progenitor cells (iNPCs). The iNPCs were then differentiated into astrocytes. Moreover, we also used a direct conversion method to generate neurons directly from the skin fibroblasts. Using these methods and performing molecular and functional analyses of the different cell types, we identified a novel disease mechanism not previously described. While IRF2BPL is localized in the nucleus in healthy individuals, we found that several patient cell lines displayed mis-localization in the form of aggregates in the cytoplasm. Moreover, the patient astrocytes also showed abnormal mitochondrial ATP-linked respiration levels and caused neuronal death in co-culture assays. Based on these observations, we tested treatment with CuATSM, a small molecule drug currently in clinical trials for ALS. We found that CuATSM successfully rescued neuronal survival and reduced ATP-linked respiration to normal levels in NEDAMSS astrocytes. We also established multiple AAV based gene therapy tools to modulate the expression of the protein and restore its normal function in vitro and potentially in vivo. In summary, we identified a novel disease mechanism involved in NEDAMSS disease and found promising therapeutic strategies that might be able to slow or halt the progression of the disease.

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity, but genetic modification of primary human NK cells has been problematic compared to the ease in which primary human T cells are transduced. Despite some progress made in using lentiviral and retroviral vectors, the efficiency of viral transduction in NK cells remains relatively low. Additionally, using these viral vectors results in random integration events which can potentially affect the safety or efficacy of transduced immune cells. Recently, we described an efficient method for gene targeting in primary human NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined that approach with Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus, and optimized transduction efficiency using various AAV pseudotypes, single-stranded (ss) or self-complementary (sc) with a wide variety of homology arms, and homology repair (HR) andor non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For initial optimization we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms for HR resulted in optimal transduction efficiency. To validate the process for generating large numbers of CAR-NK cells needed for clinical application, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered by the transgene, and the NK cells maintained high CAR expression (mean 68% CAR+) during expansion. The expanded CD33-CAR-NK cells showed enhanced activation and antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML). Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome and low off-target insertion. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

Noise-induced hearing loss (NIHL) is one of the most prevalent disabilities for which effective therapeutic treatment is currently lacking. Auditory injury caused by excessive or constant noise exposure damages the sensory elements of the ear, leading to metabolic or mechanical damage to hair cells and subsequent degeneration of spiral ganglion neurons (SGNs). To improve hearing in the NIHL patient population, maintenance and regeneration of inner hair cell-SGN synapses is critical. Although effective compounds for the treatment of NIHL have been suggested, challenges in delivery of the compounds to the inner hair cells (IHCs) hinder translation of these therapies to a clinical setting. Current routes of administration are ineffective in reaching IHCs or are invasive and may damage the cochlea. In this study, we examined less invasive delivery routes, via intrathecal (cerebrospinal fluid delivery) or intravenous injection, to administer adeno-associated virus serotype 9 (AAV9) expressing green fluorescent protein (GFP) to target IHCs and SGNs. To induce auditory injury, mice were exposed to 100 dB sound pressure level (SPL) octave band noise for 2 hours. Mice were then injected at various time points post noise injury to determine if noise exposure influenced targeting efficiency. Three weeks post injection, cochleae were processed and analyzed for GFP expression using immunofluorescence. Our results demonstrate that intravenous delivery was not successful in targeting of IHCs or SGNs at any time point measured. However, intrathecally injected AAV9 was highly effective in targeting cells in both healthy and noise-damaged cochleae at multiple time points post-noise exposure. We conclude that gene therapy techniques can be utilized to efficiently deliver therapeutic transgenes to the cochlea using cerebrospinal fluid as a delivery route. These findings determine a novel route of delivery and open a new avenue of therapeutic treatment for auditory injury and cochlear disorders.

Batten Disease is a fatal, lysosomal storage disorder characterized by cognitive and motor deficits, vision impairments, and seizures. Loss of vision is a hallmark of 10 of the 13 Batten Disease subtypes. Our group has pioneered AAV9 gene therapy treatments achieving widespread transduction of the brain and spinal cord. Two clinical trials are currently ongoing at Nationwide Children’s Hospital delivering this vector via cerebrospinal fluid (CSF) to the brain and spinal cord for treatment of the lethal neurodegenerative aspects of Batten Disease. However, AAV9 transduction of the retina after CSF delivery is limited and there is a critical need for a solution that prevents vision loss and further improves quality of life for Batten Disease patients. Similar to most genetic ocular diseases, photoreceptor degeneration is the most commonly cited pathology in patients. However, recent studies suggest that in some subtypes of Batten Disease, expression must be rescued also within the deeper layers of the retina that are difficult to reach with therapeutic vectors (inner nuclear layer, INL). We performed single cell RNA sequencing of mice and non-human primate retinas in collaboration with Dr. Fischer (OSU) and concluded that Batten Disease vision specific gene therapy needs to target a wide range of cells, including the INL, which is a major challenge for translation of a vision specific therapy to the clinic. We have recently discovered that administration of neuraminidase (NA), a sialidase enzyme, prior to or in combination with AAV9.GFP, drastically increases transduction throughout the murine C57Bl/6 retina including the INL and all the way through to the photoreceptor layer. Our preliminary data indicates GFP expression in almost all, if not all, retinal cell types using this method. Importantly, we have confirmed successful targeting of INL bipolar cells, a notoriously difficult cell-type to transduce, and up to 40% increase in Muller glia transduction. Preliminary histological examination indicates no damage or alterations in retinal integrity. While additional testing in large animal models is required and scheduled to occur in a WT pig model in Autumn 2020, this remarkable discovery suggests that it may now be possible to target every cell-type of the retina with a single AAV vector. This is especially important in regard to treatment of Batten Disease but has many additional implications. Current retinal therapies that require a more invasive subretinal delivery to photoreceptors could now opt for the safer intravitreal delivery strategy. In addition, this strategy would be highly useful in the field of optogenetics, where investigators continue to struggle to find an efficient way to express light-sensitive opsins in cells of the INL to restore sight in individuals that have already lost their photoreceptors.

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (ICV)-delivered self-complementary AAV9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well-tolerated, resulting in robust transgene expression throughout the brain and spinal cord from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and completely restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. These results demonstrate, by far, the most successful rescue reported in an animal model of CLN8 disease, and supports gene therapy as a promising therapeutic strategy for this disorder.

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (i.c.v.) delivered self-complementary adeno-associated virus serotype 9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well tolerated, resulting in robust transgene expression throughout the CNS from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. While it is unclear whether some of these behavioral improvements relate to preserved visual function, improvements in learning/memory, or other central or peripheral benefits, these results demonstrate, by far, the most successful degree of rescue reported in an animal model of CLN8 disease, and they support further development of gene therapy for this disorder.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6nclf), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6nclf mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration., Graphical Abstract, Recently, the benefits of AAV9 gene therapy were reported in a mouse model of CLN6-Batten disease, a rare neuropediatric disease. Here, the authors show that intracerebroventricularly delivered AAV9 prevents disease pathology in the visual centers of the brain, expresses in the retina, and partially preserves vision in Cln6nclf mice.

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials., Development of off-target effect-free AAV9-shRNA-mediated SOD1 silencing in an ALS mouse model, with reliable biomarkers to discern sufficient target engagement and correct dosing.

Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease.Fifteen patients with SMA1 received a single dose of intravenous adeno-associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×10As of the data cutoff on August 7, 2017, all 15 patients were alive and event-free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. In the high-dose cohort, a rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone.In patients with SMA1, a single intravenous infusion of adeno-associated viral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952 .).

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. We demonstrated that expansion of NK cells on feeder cells (CSTX002) expressing membrane-bound IL21 increases expression of HDR-related genes and provides optimum biological condition for targeted gene insertion. For proof-of-concept, we successfully generated stable mCherry-expressing primary NK cells (up to 89% mCherry+) and determined that sc vectors with 300bp homology arms were optimal. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ), which continued to show robust expansion on CSTX002 and stably maintained their CAR expression. This resulted in CAR-NK-cells of high number and purity (mean 68% CAR+) that demonstrated enhanced antileukemic activity against acute myeloid leukemia (AML) cell lines. This efficient method for site-directed insertion of genetic materials into primary NK cells has broad potential for fundamental discovery and therapeutic applications. Keywords: CRISPR, NK, Cas9/RNP, AAV6, CRISPaint, HR, CD33CAR-NK Figure Disclosures Naeimi Kararoudi: Kiadis Pharma Netherlans B.V: Patents & Royalties. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3–5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1G93Arat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1G93Arats through targeted delivery of AAV9–SOD1–shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease.

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Oligodendrocytes have recently been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). Here we show that, in vitro, mutant superoxide dismutase 1 (SOD1) mouse oligodendrocytes induce WT motor neuron (MN) hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls and patients with sporadic and familial ALS, using two different reprogramming methods. All ALS oligodendrocyte lines induced MN death through conditioned medium (CM) and in coculture. CM-mediated MN death was associated with decreased lactate production and release, whereas toxicity in coculture was lactate-independent, demonstrating that MN survival is mediated not only by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in MN rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, whereas it was ineffective in differentiated oligodendrocytes. In fact, early SOD1 knockdown rescued lactate impairment and cell toxicity in all lines tested, with the exclusion of samples carrying chromosome 9 ORF 72 (C9orf72) repeat expansions. These did not respond to SOD1 knockdown nor did they show lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type, some damage might be irreversible. In addition, we demonstrate that patients with C9ORF72 represent an independent patient group that might not respond to the same treatment.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

Introduction: Malignant glioma remains one of the most devastating diseases despite recent advances in therapies. Standard of care for malignant gliomas consists of maximal surgical resection, followed by external radiation with concurrent chemotherapy with temozolomide. Unfortunately, tumor recurrence is essentially inevitable and most of the therapies have only limited effects on the survival of patients experiencing tumor recurrence or progression. Emerging evidence has indicated that Brain Tumor Stem Cells (BTSC) can be the specific target of therapy and that elimination of BTSC will most likely result in the growth arrest of the entire tumor mass and the prevention of recurrence. The current therapies, however, preferentially target non-stem tumor cells. Thus, identification of BTSC-specific drugs is highly recommended in order to eradicate the entire tumor cells. To discover the prospective compounds from a large number of candidates efficiently, we have developed here the screening system for BTSC-targeting chemotherapeutic agents. Candidate compounds are screened in a stepwise manner based on their inhibitory potential against BTSC by using several in vitro and in vivo preclinical assays. Being different from the conventional compound discovery procedure, we combine multiple assays aiming at detection of the drugs directly targeting BTSC. Thus, this system functions as a rational agent-prioritization process essential for future progress in developing effective novel cancer treatments. Experimental Approach: We use several human BTSC cultures derived from surgical specimens. These samples have been confirmed to be clonogenic and multipotent in culture, and tumorigenic by xenotransplantation in immunodeficient mice brains. To assess the effect of compounds, the initial step is to use clonogenic assay, cell survival and migration assays with slice cultures of mouse brains, and xenotransplantation of pretreated BTSC. Potential compounds are then processed to the in vivo efficiency and efficacy validation assays using the following animal models, 1. BTSC-derived mouse brain tumor model: patient-derived BTSC transplanted in the mouse brain, 2. Genetically-engineered spontaneous mouse brain tumor model, and 3. Subcutaneous xenograft model of surgical specimens of brain tumor tissues: tumor tissue from patient directly transplanted subcutaneously into immunodeficient mice. Effect of the treatment on tumor progression is monitored by several imaging modalities including microMRI, SPECT/CT, and PET/CT. We also carry out pharmacokinetics profiling to determine the serum levels of the tested compounds and the maximum tolerated dose evaluation to determine their toxicities in comparison to their antitumor activities. In this presentation, we describe the strengths and limitations of each of our assays and also our data with the leading compounds from the previous screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5504.