Lorelei Stoica

Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

Adeno-associated virus–delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno‐associated virus (AAV)‐mediated RNAi gene therapy for ALS.

AAV delivered artificial microRNA extends survival and delays paralysis in an Amyotrophic Lateral Sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno‐associated virus (AAV)‐mediated RNAi gene therapy for ALS.

Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic.

Gene Transfer to the CNS Using Recombinant Adeno‐Associated Virus

Recombinant adeno‐associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long‐term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections. Curr. Protoc. Microbiol. 29:14D.5.1‐14D.5.18.

Restrictive Lung Disease in the Cu/Zn Superoxide-Dismutase 1 G93A Amyotrophic Lateral Sclerosis Mouse Model

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373. Optimization of AAV-Gene Therapy for GM1-Gangliosidosis

GM1-gangliosidosis is an autosomal recessive disease caused by mutations in the GLB1 gene encoding for the lysosomal acid beta-galactosidase (βgal) enzyme. The resulting enzymatic deficiency leads to accumulation of GM1-ganglioside in neurons and eventually massive neurodegeneration. The incidence of GM1-gangliosidosis is estimated at 1:100,000-200,000 live births, and there is currently no available treatment. Intracranial delivery of recombinant adeno-associated virus (AAV) vectors has been shown to be highly effective in animal models of this disease. The choice of injection sites and promoters driving transgene expression are important parameters to translate into clinical trials a safe and effective AAV-based therapy for GM1 gangliosidosis. We performed a study using AAVrh10 vectors to determine the safest and most effective promoter and delivery route combination for AAVrh10-βgal gene therapy in a GM1-mouse model. We used a combination of two direct intracranial injection site - bilateral thalamic and deep cerebral nuclei infusions (Th+DCN), or bilateral thalamic and CSF infusion into one cerebral lateral ventricle (Th+ICV). We tested three different promoters of varying strengths. The CBA promoter, composed of the chicken beta actin promoter with an enhancer element and an artificial intron; the CBi, identical to the previous promoter but lacking the enhancer element; the CB, the basic chicken beta-actin promoter without an enhancer or intron. All vectors were delivered at a total dose of 4E9 (Th+DCN) or 1E10 (Th+ICV) vector genomes. At four weeks post injection, we analyzed the brain histologically, for beta-galactosidase expression and decrease in lysosomal storage. Additionally, we analyzed the enzyme activity of beta-galactosidase, as well as the total GM1-gangliosidosis storage by enzymatic assays and LC-MS/MS mass spectrometry, respectively. The AAVrh10-CBA-βgal vector restored GM1 ganglioside levels to normal, and generated the highest βgal activity of all three vectors. Additionally, the distribution was more extensive for the AAVrh10-CBA vector, showing distribution to the frontal cortex, while for the other vectors the enzyme was mostly restricted to the injection site and the immediate area. However, at this vector dose we did not detect any apparent toxicity or cell death. Thus, we determined that the AAVrh10-CBA-βgal vector is safe and effective, and thus appropriate to carry out long-term therapeutic studies in GM1 mice toward a clinical trial.

263. A Novel Peptide-Grafted AAV Capsid Exhibits Enhanced CNS Transduction in Both Adult Mice and Cat, as Well as SOD1 Knockdown in Adult hSOD1 ALS Mice

Effective gene transfer to CNS is key for development of new therapies for neurological diseases. Relatively few natural AAV capsids cross the blood-brain barrier (BBB) upon systemic infusion and even then, transduction in adult brain after systemic delivery is limited to mainly glia and endothelium. Here we report the CNS tropism of a new AAV vector generated by genetic grafting of a small peptide into the capsid of AAV9.47, a liver-detargeted AAV9 mutant. The peptide insertion was tolerated well with no loss of packaging efficiency compared to AAV9.47 or AAV9. The scAAV-CBA-GFP vectors were infused via the tail vein in 6-8 week-old C57BL/6 mice at 5E11 vg. The new AAV vector transduced cortical and striatal neurons in the brain at high efficiency, as well as motor neurons and interneurons in the spinal cord. Significantly more vector genomes were found in the cerebrum (15-fold) and spinal cord (6-fold) compared to AAV9, while in peripheral tissues there were no significant differences. Western blot analysis of GFP expression levels confirmed the vector biodistribution results. Binding studies in parental and sialic acid-deficient CHO cells showed the interaction of the new AAV vector with terminal galactose to be unchanged compared to AAV9. The CNS tropism of the new AAV vector was evaluated in a 4 week-old cat infused with 1.26E13 vg via the carotid artery. Similar to the results in adult mice, the new AAV vector transduced cortical and striatal neurons in feline brain, and motor neurons in the feline spinal cord. These results suggest the remarkable CNS transduction properties of the new AAV may translate to higher organisms. Finally, we used the new AAV vector to knock down expression of human SOD1 in SOD1G93A mouse model of familial ALS. For these studies we used a self-complementary AAV vector encoding an artificial microRNA against human SOD1 in the 3’UTR of a CBA-GFP expression cassette. The scAAV-CBA-GFP-miR vector was infused via the tail vein in 55-day old SOD1G93A mice at 5E11 vg. At 4 weeks post-injection, there was 20% reduction in hSOD1 mRNA in the thoracic spinal cord of mice infused with the new AAV vector. The enhanced CNS targeting properties of the new AAV vector make it a promising candidate for gene therapy of neurodegenerative disorders.

341. Therapeutic Approach for SOD1-ALS Using AAV9 Delivered Artificial microRNAs

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons. This results in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About 10% of cases are inherited – typically in a dominant manner – of which twenty percent are due to mutations in the superoxide dismutase gene (SOD1). Experiments in transgenic ALS mouse models that overexpress human SOD1 have shown that decreasing levels of mutant SOD1 protein alters, and in some cases eliminates, disease progression.We postulated that silencing SOD1 expression with a micro RNA (miR) would be therapeutic in ALS. We developed a single stranded AAV9 vector encoding GFP and a miR against human SOD1, driven by a CBA promoter, which we injected into the SOD1G93A ALS mouse at postnatal day one. Each mouse received 2ml into each lateral ventricle, for a total vector dose of 4e10 vector genomes.At four weeks post injection, hSOD1 mRNA was reduced by almost 50% at all three levels of the spinal cord. Transduction was visible in both motor neurons and astrocytes in the spinal cord as well as neurons in layer V of the motor cortex. Transduced cells, assessed by GFP RNA FISH, had a decrease in hSOD1 mRNA. This translated into a 50% extension in median survival of treated mice (206 days) compared to untreated (135 days) mice.To assess neuromuscular health during the duration of the experiment, we performed motor unit number estimates (MUNEs) and needle electromyography (EMG). Treated mice had mild or no muscle denervation as opposed to the severe denervation seen in untreated SOD1. In fact, the treated mice did not develop paralysis but instead had to be euthanized due to weight loss and a hunched posture.We also assessed neuropathology in the spinal cord and nerves in untreated and treated animals, at their respective endpoints. The treated mice showed no spinal cord motor neuron loss, while the untreated mice lost the majority of the motor neurons. The axonal integrity of the lumbar ventral roots was also improved in treated animals. Furthermore, there was no axonal degeneration in the sciatic nerves of the treated animals when analyzed at 120 days, the endpoint of untreated mice. Lastly, treated animals show delayed onset of astrogliosis and microgliosis, as observed by IHC for inflammatory markers GFAP and Iba1, and confirmed by RT-qPRC for genes upregulated in inflammation.In conclusion, we were successful at extending the lifespan of the SOD1G93A mouse by 50% with our high dose neonatal AAV9-miR, and our treated animals remain ambulatory and active until the humane endpoint with minimal or no signs of paralysis.

UNIT 14D.5 Gene Transfer to the CNS Using Recombinant Adeno-Associated Virus

Recombinant adeno‐associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long‐term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections. Curr. Protoc. Microbiol. 29:14D.5.1‐14D.5.18.