- Email: [email protected]
617. Identification of Novel AAV Capsid Variants with Enhanced Tropism for the Canine Outer Retina Following Intravitreal Delivery
Delivery Questions for Neural Applications Collectively, these studies reflect the simplicity and efficacy of this commercially available technology in presenting a cost-effective and advantageous avenue for screening and validating new targeted nucleic acid therapies.
for disease correction. The potential impact on clinical practice in the field of LSD is high since, if these results extend to larger animal disease models, nonhuman primates, and human subjects, the largest current barriers to health for affected patients would be circumvented.
615. Choroid Plexus-Targeted Viral Gene Therapy for Lysosomal Storage Diseases
616. Tailored Expression of a Transgene to Specific Cell Types in the Central Nervous System After Peripheral Injection of AAV9
Eun-Young Choi1, Shih-hsin Kan2, S. Q. Le2, Patricia I. Dickson2, Stephen G. Kaler1 1 NIH, Bethesda, MD, 2Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA Lysosomal storage diseases (LSDs) represent a category of approximately 60 inherited neurometabolic illnesses with considerable morbidity and mortality in children and adults. The burden of LSD is high due to the chronic progressive decline in the neurocognitive function of affected individuals that profoundly limits their societal integration. Current therapy for LSDs has focused on recombinant enzyme replacement therapy (ERT), which while promising, is not ideal for numerous reasons, including the short half-lives of ERT with weekly or monthly administration, potential immune responses against ERT may also reduce the effectiveness for the treatment and inefficient passage through the blood-brain barrier. The choroid plexuses are vascularized structures that project into the cerebrospinal fluid (CSF) and feature specialized polarized epithelia derived from neuroectoderm that are post-mitotic, i.e., do not undergo turnover, and produce CSF by transporting water and ions into the brain ventricles. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time administration of a recombinant AAV vector with selective tropism for choroid plexus (e.g., serotype 5) would be an attractive strategy for long-term treatment of LSDs. Potentially this approach would result in steady secretion of the missing enzyme into the CSF, which normally carries molecules throughout the ventricular system into the subarachnoid space, and ultimately deliver enzyme to the entire brain. The cross-correction phenomenon in many LSDs would provide a further advantage. To evaluate this hypothesis in preclinical animal models, we chose two prototypical LSDs, α-mannosidosis and mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B syndrome). We cloned the respective cDNAs (human (h) LAMAN and NAGLU) into rAAV shuttle plasmids and generated high titer of rAAV5 expressing the enzymes. We administered viral particles to the lateral cerebroventricles sof homozygous mutant mice on day 2 or 3 of life at doses of 5 x 109 or 5 x 1010 vector genomes. In the LAMAN deficient mice, we documented dose-dependent transduction and hLAMAN mRNA expression confined to the choroid plexuses of rAAV5-treated animals. Brain biochemical analyses at 1, 2 and 6 months post-treatment documented sustained, highly statistically significant increases of LAMAN enzyme activity in the brain globally (olfactory bulb, cerebral cortex, cerebellum, brainstem). By 8 months of age, untreated mutant mice showed prominent lysosomal vacuoles in hippocampal neurons, in contrast to rAAV5-hLAMAN treated mutants for which brain histopathology was comparable to wild-type. Lysosomal associated membrane protein 1 (Lamp1) levels were normalized in AAV5-hLAMAN treated mutant brain. In MPS IIIB mutant mice, levels of NAGLU enzyme activity were 2-8 fold higher in brain sections six weeks after rAAV5-hNAGLU treatment compared to normal controls. β-Hexosaminidase activity, which is elevated in MPS IIIB, was reduced to heterozygote carrier levels. Tissue evaluations by immunohistochemistry showed robust hNAGLU expression in the choroid plexus epithelia. Lamp1 expression was significantly reduced in hippocampus and frontal cortex of treated mice. The approach outlined herein challenges existing treatment modalities for LSDs, by exploiting the ability of choroid plexus-targeted gene therapy to restore missing or defective lysosomal enzymes at concentrations and distributions in CSF suitable S244
Eloise Hudry, Jonathan Dashkoff, Paul Lerner, Shuko Takeda, Nhi Truong, Casey Maguire, Bradley T. Hyman Massachusetts General Hospital -Harvard Medical School, Charlestown, MA Because of their efficient tropism for the neural tissue and their well-established safety and tolerability profile, adeno-associated vectors (AAV) hold great potential to express therapeutic genes in the context of various neurodegenerative disorders. Importantly, the characterization of novel AAV serotypes (AAV9; rhAAV10, etc⋯) that can cross the blood brain barrier after intravenous delivery now offer an opportunity for non-invasive delivery to the brain. However, in absence of a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in various cell types of the nervous system as well as in other organs. In order to refine this approach, the present study characterizes the transduction profiles of newly engineered self-complement AAV9 (scAAV9) expressing the Green Fluorescent Protein (GFP) either under an astrocyte (GFA’) or neuronal (Synapsin, Syn) promoter, after a single injection in the lateral tail vein of adult mice (5x10e11vg/animal). We report that our scAAV9-GFA’-GFP and scAAV9-Syn-GFP respectively led to robust and long-lasting transduction of astrocytes (10%) and neurons (8%) throughout the entire cerebral tissue, in the absence of aberrant expression leakage in other cell types of the brain or in the liver. GFP positive excitatory as well as inhibitory neurons could be observed in the CNS, while motor neurons were also efficiently transduced in the PNS. In addition, both activated (GFAP positive) and resting astrocytes expressed the reporter gene. Quantitative stereological assessment demonstrated an increase in reactive astrocytes after transduction with scAAV9 but did not lead to any obvious detrimental phenotype in any animals. These data suggest the potential interest of tailoring AAV to drive expression of therapeutic genes specifically in astrocytes or neurons of the CNS after intravenous delivery, thus preventing adverse side-effects associated with peripheral expression.
617. Identification of Novel AAV Capsid Variants with Enhanced Tropism for the Canine Outer Retina Following Intravitreal Delivery
Leah C. Byrne1, Meike Visel2, Valerie Dufour3, Felipe Pompeo Marinho3, Gustavo D. Aguirre3, William A. Beltran3, John G. Flannery2, David V. Schaffer2 1 Department of Clinical Studies, School of Veterinary Medicine/ Helen Wills Neuroscience Institute, University of Pennsylvania/ University of California Berkeley, Berkeley, CA, 2Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, 3Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA The delivery of AAV vectors across physical barriers to target the outer retina is key to the success of retinal gene therapies. Subretinal injection of AAV vectors is the most common approach to deliver genes to photoreceptors and RPE; however, this route of administration presents surgical challenges and potential complications, as well as mediates transfer to only a limited region of the retinal surface. Intravitreal administration may represent a safer way of targeting larger areas of the outer retina; physical barriers such Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Delivery Questions for Neural Applications as the internal limiting membrane significantly limit efficiency of natural AAVs through this route. In a prior murine proof of concept study, we showed that directed evolution is promising method for engineering new AAV capsid variants with enhanced permissivity for the outer retina. Here, we show results from a screen performed in the canine retina, a large animal model with eye structure and size similar to the human eye. A library of >107 distinct capsid variant AAV vectors was injected intravitreally into the eye of a wildtype dog. A subset of the AAV vectors injected was recovered from samples of outer retina by PCR amplification. Isolated variants were then pooled and re-injected in additional wildtype canine eyes, and following 5 total rounds of selection the pool had converged to a relatively small number of variants. High throughput sequencing was used to characterize the convergence of variants over all rounds of selection. The final candidate variants were then evaluated by packaging a GFP reporter controlled by a ubiquitous CAG promoter. Retinal distribution and cellular tropism of the variants was examined by cSLO (AF mode) imaging, histology and immunocytochemistry. Nineteen variants were identified as candidate vectors for targeting the canine outer retina. Intravitreal injection of a mixture of equal amounts of these variants carrying GFP fused to a barcode resulted in efficient targeting of all retinal layers including photoreceptors and RPE in 6 eyes of 3 wildtype dogs. Thus, directed evolution performed in a large animal model, with an eye structure similar to that of humans, resulted in new AAV capsid variants with improved abilities to bypass significant structural barriers and efficiently transduce the outer retina. High throughput sequencing allowed for identification of promising variants and monitoring of convergence. These vectors show promise for gene therapy delivery via the intravitreal route of administration, potentially providing a safer approach for targeting a larger area of outer retina than currently achieved by the more traumatic and focal subretinal injection method.
618. Directional Transduction of AAV-5 Vectors in the NHP Brain
Bas Blits1, Lluis Samaranch2, John Bringas2, Waldy San Sebastian2, Krystoff Bankiewicz2, Harald Petry3 1 Neurobiology Research, uniQure, Amsterdam, Netherlands, 2 Neurological Surgery, University of California San Francisco, San Francisco, CA, 3Research, uniQure, Amsterdam, Netherlands This study was performed to investigate the delivery of AAV vectors into the putamen and thalamus of nonhuman primates. The vectors were delivered to the targeted brain regions by MRI-guided convection enhanced delivery. Special attention was given to the analysis of axonal transport and levels of transduction. Recombinant AAV is an excellent candidate for delivery of therapeutic molecules to the central nervous system to target neurodegenerative diseases. UniQure has succeeded in developing a proprietary platform manufacturing technology that allows safe, effective, cGMPcompliant, economically feasible and commercially scalable manufacturing of AAV. UniQure’s novel approach is based on the use of a combination of recombinant baculoviruses and insect cells. Using our production platform, two AAV stocks encoding GDNF or GFP were generated. At eight weeks following infusion into the thalamus, for instance, massive transduction of the thalamus, cortex, striatum and substantia nigra was observed indicating both anterograde and retrograde transduction. At the site of injection, transduction was both glial and neuronal, whereas off site transduction was mainly neuronal. Following injection of a lower AAV volume into the putamen, transduction was limited to areas within the putamen and substantia nigra suggesting only anterograde transport of viral particles. These data indicate a dose dependent anterograde or retrograde transport mechanism. This data set confirms that production of AAV using the (scalable) baculovirus-based platform results in an effective vector Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
that is able to mediate expression patterns that can be used to develop an AAV-mediated therapeutic strategy to treat neurodegenerative diseases.
619. AAV9 Transduction Is Similar in Adult and Aged Mouse Brains Following Intraparenchymal Injection
Sara E. Gombash1, Julie A. Fitzgerald1, Christopher J. Cowley1, Mitchell G. Neides1, Emily Armstrong1, Diana M. Norden2, Jonathan P. Godbout1, Kevin D. Foust1 1 Neuroscience, Ohio State University, Columbus, OH, 2Physical Therapy, Ohio State University, Columbus, OH
We previously reported that systemic injection of AAV9 in aged mice (P550) results in significantly fewer transduced CNS cells compared to adult mice (P49). This was not due to the presence of neutralizing antibodies in the aged mice. To determine whether aging in the brain and/or the periphery is responsible for this reduction, we quantified CNS transduction following stereotaxic brain injection in aged or adult mice. Aged male Balb/c mice (n = 6, P550) and adult mice (n = 7, P50) received direct injections into the striatum and ipsilateral hippocampus with scAAV9-CB expressing green fluorescent protein or scAAV9-CB expressing glial cell line derived neurotrophic factor (GFP or GDNF). All mice were euthanized three weeks post-injection. Brains of aged and adult mice injected with scAAV9-CB-GFP were sliced and processed for near-infrared imaging to quantify vector spread and GFP intensity. GFP expression was similar in directly injected aged and adult brains. Vector spread along the rostrocaudal axis was approximately 2 mm from either the striatal or hippocampal injection site and did not vary with age. Brains of mice directly injected with scAAV9-CB-GDNF were hemisected and processed for ELISA to quantify GDNF protein levels. GDNF levels in the striatum and hippocampus reached 2x those of uninjected controls and no differences in GDNF expression were detected between aged and adult injected mice. Together, we found that AAV9 CNS transduction is similar in aged and adult mouse brains following direct injection. These results suggest that reduced CNS transduction in aged mice following AAV9 systemic injection is due to alterations in the periphery. Future studies include investigation of altered viral blood brain barrier penetration and viral retention within peripheral tissues of aged mice.
620. Use of Transgenic Mice to Quantify Transduction Efficiency and Specificity of Novel AAV Vectors in Retina
Miranda L. Scalabrino, K. Tyler McCullough, Shreyasi Choudhury, James J. Peterson, Seok-Hong Min, Sanford L. Boye, Shannon E. Boye Ophthalmology, University of Florida, Gainesville, FL Modifications to surface-exposed residues of recombinant Adenoassociated virus (AAV), incorporation of novel regulatory elements into the vector cassette, alternative injection routes, and combinations thereof are being explored as strategies to enhance gene delivery to retina. We previously showed that intravitreal injection of a novel AAV capsid was capable of transducing all neuronal layers of the retina, including photoreceptors (PRs). We also demonstrated that this capsid, when combined with a novel promoter (“Ple155”) was capable of driving therapeutic nyctalopin (Nyx) expression exclusively in ON bipolar cells (ON BCs) and partially restoring retinal function to a mouse model of complete Congenital Stationary Night Blindness (CSNB1) following early (postnatal day 2) treatment. In order to achieve more efficient transduction of PRs and ON BCs, further modifications will be required. Methods with which to quantify relative transduction efficiencies of novel variants in vivo are S245
for disease correction. The potential impact on clinical practice in the field of LSD is high since, if these results extend to larger animal disease models, nonhuman primates, and human subjects, the largest current barriers to health for affected patients would be circumvented.
615. Choroid Plexus-Targeted Viral Gene Therapy for Lysosomal Storage Diseases
616. Tailored Expression of a Transgene to Specific Cell Types in the Central Nervous System After Peripheral Injection of AAV9
Eun-Young Choi1, Shih-hsin Kan2, S. Q. Le2, Patricia I. Dickson2, Stephen G. Kaler1 1 NIH, Bethesda, MD, 2Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA Lysosomal storage diseases (LSDs) represent a category of approximately 60 inherited neurometabolic illnesses with considerable morbidity and mortality in children and adults. The burden of LSD is high due to the chronic progressive decline in the neurocognitive function of affected individuals that profoundly limits their societal integration. Current therapy for LSDs has focused on recombinant enzyme replacement therapy (ERT), which while promising, is not ideal for numerous reasons, including the short half-lives of ERT with weekly or monthly administration, potential immune responses against ERT may also reduce the effectiveness for the treatment and inefficient passage through the blood-brain barrier. The choroid plexuses are vascularized structures that project into the cerebrospinal fluid (CSF) and feature specialized polarized epithelia derived from neuroectoderm that are post-mitotic, i.e., do not undergo turnover, and produce CSF by transporting water and ions into the brain ventricles. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time administration of a recombinant AAV vector with selective tropism for choroid plexus (e.g., serotype 5) would be an attractive strategy for long-term treatment of LSDs. Potentially this approach would result in steady secretion of the missing enzyme into the CSF, which normally carries molecules throughout the ventricular system into the subarachnoid space, and ultimately deliver enzyme to the entire brain. The cross-correction phenomenon in many LSDs would provide a further advantage. To evaluate this hypothesis in preclinical animal models, we chose two prototypical LSDs, α-mannosidosis and mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B syndrome). We cloned the respective cDNAs (human (h) LAMAN and NAGLU) into rAAV shuttle plasmids and generated high titer of rAAV5 expressing the enzymes. We administered viral particles to the lateral cerebroventricles sof homozygous mutant mice on day 2 or 3 of life at doses of 5 x 109 or 5 x 1010 vector genomes. In the LAMAN deficient mice, we documented dose-dependent transduction and hLAMAN mRNA expression confined to the choroid plexuses of rAAV5-treated animals. Brain biochemical analyses at 1, 2 and 6 months post-treatment documented sustained, highly statistically significant increases of LAMAN enzyme activity in the brain globally (olfactory bulb, cerebral cortex, cerebellum, brainstem). By 8 months of age, untreated mutant mice showed prominent lysosomal vacuoles in hippocampal neurons, in contrast to rAAV5-hLAMAN treated mutants for which brain histopathology was comparable to wild-type. Lysosomal associated membrane protein 1 (Lamp1) levels were normalized in AAV5-hLAMAN treated mutant brain. In MPS IIIB mutant mice, levels of NAGLU enzyme activity were 2-8 fold higher in brain sections six weeks after rAAV5-hNAGLU treatment compared to normal controls. β-Hexosaminidase activity, which is elevated in MPS IIIB, was reduced to heterozygote carrier levels. Tissue evaluations by immunohistochemistry showed robust hNAGLU expression in the choroid plexus epithelia. Lamp1 expression was significantly reduced in hippocampus and frontal cortex of treated mice. The approach outlined herein challenges existing treatment modalities for LSDs, by exploiting the ability of choroid plexus-targeted gene therapy to restore missing or defective lysosomal enzymes at concentrations and distributions in CSF suitable S244
Eloise Hudry, Jonathan Dashkoff, Paul Lerner, Shuko Takeda, Nhi Truong, Casey Maguire, Bradley T. Hyman Massachusetts General Hospital -Harvard Medical School, Charlestown, MA Because of their efficient tropism for the neural tissue and their well-established safety and tolerability profile, adeno-associated vectors (AAV) hold great potential to express therapeutic genes in the context of various neurodegenerative disorders. Importantly, the characterization of novel AAV serotypes (AAV9; rhAAV10, etc⋯) that can cross the blood brain barrier after intravenous delivery now offer an opportunity for non-invasive delivery to the brain. However, in absence of a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in various cell types of the nervous system as well as in other organs. In order to refine this approach, the present study characterizes the transduction profiles of newly engineered self-complement AAV9 (scAAV9) expressing the Green Fluorescent Protein (GFP) either under an astrocyte (GFA’) or neuronal (Synapsin, Syn) promoter, after a single injection in the lateral tail vein of adult mice (5x10e11vg/animal). We report that our scAAV9-GFA’-GFP and scAAV9-Syn-GFP respectively led to robust and long-lasting transduction of astrocytes (10%) and neurons (8%) throughout the entire cerebral tissue, in the absence of aberrant expression leakage in other cell types of the brain or in the liver. GFP positive excitatory as well as inhibitory neurons could be observed in the CNS, while motor neurons were also efficiently transduced in the PNS. In addition, both activated (GFAP positive) and resting astrocytes expressed the reporter gene. Quantitative stereological assessment demonstrated an increase in reactive astrocytes after transduction with scAAV9 but did not lead to any obvious detrimental phenotype in any animals. These data suggest the potential interest of tailoring AAV to drive expression of therapeutic genes specifically in astrocytes or neurons of the CNS after intravenous delivery, thus preventing adverse side-effects associated with peripheral expression.
617. Identification of Novel AAV Capsid Variants with Enhanced Tropism for the Canine Outer Retina Following Intravitreal Delivery
Leah C. Byrne1, Meike Visel2, Valerie Dufour3, Felipe Pompeo Marinho3, Gustavo D. Aguirre3, William A. Beltran3, John G. Flannery2, David V. Schaffer2 1 Department of Clinical Studies, School of Veterinary Medicine/ Helen Wills Neuroscience Institute, University of Pennsylvania/ University of California Berkeley, Berkeley, CA, 2Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, 3Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA The delivery of AAV vectors across physical barriers to target the outer retina is key to the success of retinal gene therapies. Subretinal injection of AAV vectors is the most common approach to deliver genes to photoreceptors and RPE; however, this route of administration presents surgical challenges and potential complications, as well as mediates transfer to only a limited region of the retinal surface. Intravitreal administration may represent a safer way of targeting larger areas of the outer retina; physical barriers such Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Delivery Questions for Neural Applications as the internal limiting membrane significantly limit efficiency of natural AAVs through this route. In a prior murine proof of concept study, we showed that directed evolution is promising method for engineering new AAV capsid variants with enhanced permissivity for the outer retina. Here, we show results from a screen performed in the canine retina, a large animal model with eye structure and size similar to the human eye. A library of >107 distinct capsid variant AAV vectors was injected intravitreally into the eye of a wildtype dog. A subset of the AAV vectors injected was recovered from samples of outer retina by PCR amplification. Isolated variants were then pooled and re-injected in additional wildtype canine eyes, and following 5 total rounds of selection the pool had converged to a relatively small number of variants. High throughput sequencing was used to characterize the convergence of variants over all rounds of selection. The final candidate variants were then evaluated by packaging a GFP reporter controlled by a ubiquitous CAG promoter. Retinal distribution and cellular tropism of the variants was examined by cSLO (AF mode) imaging, histology and immunocytochemistry. Nineteen variants were identified as candidate vectors for targeting the canine outer retina. Intravitreal injection of a mixture of equal amounts of these variants carrying GFP fused to a barcode resulted in efficient targeting of all retinal layers including photoreceptors and RPE in 6 eyes of 3 wildtype dogs. Thus, directed evolution performed in a large animal model, with an eye structure similar to that of humans, resulted in new AAV capsid variants with improved abilities to bypass significant structural barriers and efficiently transduce the outer retina. High throughput sequencing allowed for identification of promising variants and monitoring of convergence. These vectors show promise for gene therapy delivery via the intravitreal route of administration, potentially providing a safer approach for targeting a larger area of outer retina than currently achieved by the more traumatic and focal subretinal injection method.
618. Directional Transduction of AAV-5 Vectors in the NHP Brain
Bas Blits1, Lluis Samaranch2, John Bringas2, Waldy San Sebastian2, Krystoff Bankiewicz2, Harald Petry3 1 Neurobiology Research, uniQure, Amsterdam, Netherlands, 2 Neurological Surgery, University of California San Francisco, San Francisco, CA, 3Research, uniQure, Amsterdam, Netherlands This study was performed to investigate the delivery of AAV vectors into the putamen and thalamus of nonhuman primates. The vectors were delivered to the targeted brain regions by MRI-guided convection enhanced delivery. Special attention was given to the analysis of axonal transport and levels of transduction. Recombinant AAV is an excellent candidate for delivery of therapeutic molecules to the central nervous system to target neurodegenerative diseases. UniQure has succeeded in developing a proprietary platform manufacturing technology that allows safe, effective, cGMPcompliant, economically feasible and commercially scalable manufacturing of AAV. UniQure’s novel approach is based on the use of a combination of recombinant baculoviruses and insect cells. Using our production platform, two AAV stocks encoding GDNF or GFP were generated. At eight weeks following infusion into the thalamus, for instance, massive transduction of the thalamus, cortex, striatum and substantia nigra was observed indicating both anterograde and retrograde transduction. At the site of injection, transduction was both glial and neuronal, whereas off site transduction was mainly neuronal. Following injection of a lower AAV volume into the putamen, transduction was limited to areas within the putamen and substantia nigra suggesting only anterograde transport of viral particles. These data indicate a dose dependent anterograde or retrograde transport mechanism. This data set confirms that production of AAV using the (scalable) baculovirus-based platform results in an effective vector Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
that is able to mediate expression patterns that can be used to develop an AAV-mediated therapeutic strategy to treat neurodegenerative diseases.
619. AAV9 Transduction Is Similar in Adult and Aged Mouse Brains Following Intraparenchymal Injection
Sara E. Gombash1, Julie A. Fitzgerald1, Christopher J. Cowley1, Mitchell G. Neides1, Emily Armstrong1, Diana M. Norden2, Jonathan P. Godbout1, Kevin D. Foust1 1 Neuroscience, Ohio State University, Columbus, OH, 2Physical Therapy, Ohio State University, Columbus, OH
We previously reported that systemic injection of AAV9 in aged mice (P550) results in significantly fewer transduced CNS cells compared to adult mice (P49). This was not due to the presence of neutralizing antibodies in the aged mice. To determine whether aging in the brain and/or the periphery is responsible for this reduction, we quantified CNS transduction following stereotaxic brain injection in aged or adult mice. Aged male Balb/c mice (n = 6, P550) and adult mice (n = 7, P50) received direct injections into the striatum and ipsilateral hippocampus with scAAV9-CB expressing green fluorescent protein or scAAV9-CB expressing glial cell line derived neurotrophic factor (GFP or GDNF). All mice were euthanized three weeks post-injection. Brains of aged and adult mice injected with scAAV9-CB-GFP were sliced and processed for near-infrared imaging to quantify vector spread and GFP intensity. GFP expression was similar in directly injected aged and adult brains. Vector spread along the rostrocaudal axis was approximately 2 mm from either the striatal or hippocampal injection site and did not vary with age. Brains of mice directly injected with scAAV9-CB-GDNF were hemisected and processed for ELISA to quantify GDNF protein levels. GDNF levels in the striatum and hippocampus reached 2x those of uninjected controls and no differences in GDNF expression were detected between aged and adult injected mice. Together, we found that AAV9 CNS transduction is similar in aged and adult mouse brains following direct injection. These results suggest that reduced CNS transduction in aged mice following AAV9 systemic injection is due to alterations in the periphery. Future studies include investigation of altered viral blood brain barrier penetration and viral retention within peripheral tissues of aged mice.
620. Use of Transgenic Mice to Quantify Transduction Efficiency and Specificity of Novel AAV Vectors in Retina
Miranda L. Scalabrino, K. Tyler McCullough, Shreyasi Choudhury, James J. Peterson, Seok-Hong Min, Sanford L. Boye, Shannon E. Boye Ophthalmology, University of Florida, Gainesville, FL Modifications to surface-exposed residues of recombinant Adenoassociated virus (AAV), incorporation of novel regulatory elements into the vector cassette, alternative injection routes, and combinations thereof are being explored as strategies to enhance gene delivery to retina. We previously showed that intravitreal injection of a novel AAV capsid was capable of transducing all neuronal layers of the retina, including photoreceptors (PRs). We also demonstrated that this capsid, when combined with a novel promoter (“Ple155”) was capable of driving therapeutic nyctalopin (Nyx) expression exclusively in ON bipolar cells (ON BCs) and partially restoring retinal function to a mouse model of complete Congenital Stationary Night Blindness (CSNB1) following early (postnatal day 2) treatment. In order to achieve more efficient transduction of PRs and ON BCs, further modifications will be required. Methods with which to quantify relative transduction efficiencies of novel variants in vivo are S245