Research Interests

My core research focus is to develop adeno-associated virus (AAV) gene transfer vector systems, for clinically-relevant global gene transfer to the central and peripheral nervous system. This research focus has also included preclinical studies to apply these engineered AAV systems toward treatments for neurological diseases in animal models. Currently these include Rett Syndrome, Giant Axonal Neuropathy, and Lysosomal Storage Diseases (Tay-Sachs, LINCL, INCL, AGU, and Krabbe). My future directions include 1) continued development and optimization of AAV vectors specifically tailored toward CNS applications, 2) expanding this program to include therapeutic approaches for other applicable CNS disorders, and 3) facilitating the translation of these approaches from bench to clinic.


Vector Design and Optimization for CNS Applications

A major obstacle for most gene therapy approaches for CNS diseases is a means to broadly and efficiently deliver a gene to the entire extent of the brain and spinal cord. An ideal vector and delivery approach can be used to package any number of genes to potentially treat a wide variety of diseases. Besides the goal of global CNS gene delivery, the development of reagents to deliver a transgene to specific neural cell subtypes (neurons, astrocytes, oligodendrocytes, microglia) would have value for research and therapeutic applications.

Intravenous or intra-CSF delivery approaches using an AAV serotype 9 vector achieve widespread CNS delivery in rodents, pigs (for intra-CSF), and non-human primates. We identified barriers to the intravenous approach in the non-human primate studies (see Gray et al., 2011, Molecular Therapy), and have pursued intra-CSF vector administration to overcome these barriers (see Federici et al., 2012, Gene Therapy; Gray et al., 2013, Molecular Therapy). These studies have indicated a delivery efficiency that could be appropriate for diseases where the therapeutic gene product would be secreted, such as ALS and lysosomal storage diseases (Krabbe, Batten, Tay-Sachs, AGU, etc.). The intravenous approach is still being pursued, as a way to take advantage of a blood-brain-barrier pathology that is present in many CNS diseases (such as epilepsy and Parkinson’s). Using an AAV capsid DNA shuffling and directed evolution approach, novel AAV vectors were developed that can specifically target damaged areas of the brain following intravenous injection of the vector, in a rat model for epilepsy. Also, modified-AAV9 vectors were developed that retain CNS tropism while losing tropism for peripheral organs like the heart and liver. As part of ongoing unpublished studies, we are developing AAV vectors that target specific neuronal and glial populations. Along with work to better target the AAV capsid to the CNS, we have optimized the packaged gene cassettes provide high, low, ubiquitous, or neuron-restricted gene expression.

Future Research Directions. The AAV capsid gene shuffling and directed evolution methodology has been a powerful and successful tool to generate superior AAV-based gene delivery vehicles (see Gray et al., 2010, Molecular Therapy). This approach will be used in a general sense to make more efficient and better-targeted vectors. We will also further explore the potential of this approach to tailor AAV vectors to specific diseases, to take advantage of the natural pathology of diseases to target the therapeutic genes where they are needed, as we published for an epilepsy-specific vector.

Disease Applications

The existing level of gene delivery seen in pigs and non-human primates with intrathecal AAV9 delivery has strong potential to provide therapeutic efficacy in a number of lysosomal storage diseases, where the replaced enzyme can confer cross-correction via the mannose-6-phosphate pathway and a broad therapeutic effect can be expected from a diffuse pattern of transduced cells. This approach also can successfully deliver a gene to 50-100% of spinal cord motor neurons, which has strong therapeutic implications for ALS, spinal muscular atrophy, and giant axonal neuropathy.

Giant Axonal Neuropathy (GAN) is an axonal degenerative disease that is fatal by the 3rd decade of life. It is caused by the loss of a single gene (gigaxonin), and is characterized by intermediate filament inclusion bodies, peripheral neuropathy (motor and sensory), white matter abnormalities, and later atrophy of the brainstem. A foundation called Hannah’s Hope Fund has sponsored an effort to develop and test a gene therapy approach for GAN, which is centered at UNC under my direction. This project was started in fall of 2008, and in January 2012 we discussed the preclinical data generated from our studies with the FDA in a preIND meeting, with positive feedback. Our clinical trial intentions were approved by the NIH Recombinant DNA Advisory Committee in June 2013, and we are planning to begin a gene therapy clinical trial for GAN in 2014, which would be supported by Hannah’s Hope Fund and conducted at the NIH Clinical Center in Bethesda, MD.

Epilepsy. This project is in collaboration with Thomas McCown at UNC. Novel AAV vectors were developed that can be injected intravenously and targeted very specifically to areas in the brain that are affected by the seizures. Previous published data from Dr. McCown has shown that AAV vectors delivering the galanin gene can attenuate seizures. Current and future studies are refining the vectors to increase their efficiency and to test a therapeutic gene (galanin) for efficacy in the rat seizure model.

Rett Syndrome (RTT) is a severe form of inherited mental retardation that is fatal in males (X-linked) and extremely debilitating in females. Most cases are caused by a loss of the MeCP2 gene. Introduction of a functional copy of the MeCP2 gene has potential to reverse many of the symptoms of RTT. To realize a successful RTT gene therapy, it is expected that 2 major obstacles must be overcome: 1) to provide the correct level of MeCP2 (since overexpression can be deleterious) and 2) to achieve widespread and efficient delivery of the MeCP2 gene. This research has focused on both obstacles through testing of the MeCP2 promoter in AAV/MeCP2 constructs, optimizing strategies to achieve global and efficient AAV delivery to the CNS, and engineering modified AAV capsids to increase the efficiency of gene transfer in the RTT mice. In proof-of-concept studies recently published in Molecular Therapy, we showed that delivery of AAV/MeCP2 vectors in male KO MeCP2 mice provided survival and phenotypic benefits, but our studies also identified barriers for human translation. Ongoing and future studies are aimed at engineering AAV vectors that preferentially target MeCP2-null cells over WT cells in a heterozygous RTT female. We are exploring approaches to translate RTT gene therapy to human patients.

Tay-Sachs, Krabbe, Batten, AGU, and lysosomal storage diseases in general. Generally speaking, many lysosomal storage diseases are caused by the lack a single enzyme that results in the accumulation of its substrate. These substrate accumulations become toxic, with the initial (and fatal) effects presenting in the CNS. Enzyme replacement therapies (ERTs) are being developed or are available for a number of these diseases, but this only treats the peripheral symptoms if the enzymes are not transported across the blood-brain barrier. CNS-directed gene therapy offers a way to produce the missing enzyme within the CNS to directly treat the neurological defects in these diseases. This approach has shown partial efficacy for a number of diseases by stereotaxic delivery of the therapeutic gene to the brain. The global gene delivery approach utilized in our studies (for rodents, pigs, and monkeys) offers a more effective and less invasive approach to treat these diseases. Preclinical testing is underway for Krabbe Disease, Tay-Sachs Disease, AGU, and Batten Disease (INCL and LINCL).


Sloniowski S, Fox JC, Gray SJ* (2013) Perspectives in using gene therapy for lysosomal storage diseases. Drugs of the Future, in press. [*corresponding author]

Nagabhushan Kalburgi S, Khan NN, Gray SJ* (2013) Recent gene therapy advancements for neurological diseases. Discovery Medicine, 15(81):111-9. PMID: 23449113. [*corresponding author]

Simonato M, Bennett J, Boulis NM, Castro MG, Fink DJ, Gray SJ, Lowenstein PR, Tobin AL, Vandenberghe LH, Wolfe JH, and Glorioso JC. Progress in gene therapy for neurological disorders. Nature Reviews Neuroscience, 9(5):277-91. PMID: 23670108.

Sinici I, Yonekawa S, Tkachyova I, Gray SJ, Samulski RJ, Wakarchuk W, Mark BL, Mahuran D. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside. PLOSone, in press. PMID: 23483939. PMCID: 3587417.

Gray SJ*, Nagabhushan Kalburgi S, McCown TJ, Samulski RJ (2013) Global CNS Gene Delivery and Evasion of Anti-AAV Neutralizing Antibodies by Intrathecal Vector Administration in Non-Human Primates. Gene Therapy, 20(4):450-9. PMID: 23303281. PMCID: 3618620. [*corresponding author]

Mussche S, Devreese B, Nagabhushan Kalburgi S, Bachaboina L, Fox JC, Shih HJ, Samulski RJ, Van Coster R, Gray SJ* (2013) Restoration of Cytoskeleton Homeostasis After Gigaxonin Gene transfer for Giant Axonal Neuropathy. Human Gene Therapy, 24(2):209-19. PMID:23316953. [*corresponding author] Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, Gray SJ, and Samulski RJ (2013) Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis. Molecular Therapy – Nucleic Acids, 2:e70.

Gray SJ (2013) Gene Therapy and Neurodevelopmental Disorders. Neuropharmacology, 68:136-42. PMID: 22750077.

Gadalla KKE, Bailey MES, Spike RC, Ross PD, Woodard KT, Nagabushan Kalburgi S, Bachaboina L, Deng JV, West AE, Samulski RJ, Gray SJ*, andCobb SR* (2013) Survival Benefit and Phenotypic Improvement of Male Rett Syndrome Mice Following Neonatal and Juvenile AAV9/MeCP2 Gene Transfer. Molecular Therapy, 21(1):18-30. PMID: 23011033. PMCID: 3536818. [*co-corresponding and -senior authors]

Bowles DE, McPhee SWJ, Li C, Gray SJ, Samulski JJ, Camp AS, Li J, Wang B, Monahan PE, Rabinowitz JE, Grieger JC, Govindasamy L, Agbandje-McKenna M, Xiao X, Samulski RJ (2012) Phase 1 Gene Therapy for Duchenne Muscular Dystrophy using a Designer AAV Vector. Molecular Therapy, 20(2):443-55. PMID: 22068425. PMCID: 3277234.

Lentz TB, Gray SJ, Samulski RJ (2012) Viral Vectors for Gene Delivery to the Central Nervous System. Neurobiology of Disease, 48(2):179-88.PMID 22001604. PMCID: 3293995.

Federici T, Taub JS, Baum GR, Gray SJ, Grieger JC, Matthews K, Handy C, Passini MA, Samulski RJ, and Boulis NM (2012) Robust Spinal Motor Neuron Transduction Following Intrathecal Delivery of AAV9 in Pigs. Gene Therapy, 19(8):852-9. PMID: 21918551.

Gray SJ, Choi VW, Asokan A, Haberman RA, McCown TJ, and Samulski RJ (2011) Production of Recombinant Adeno-Associated Viral Vectors and Use in In Vitro and In Vivo Administration. Current Protocols in Neuroscience, Chapter 4:Unit4.17. PMID: 21971848.PMCID: 3209619.

Li C, Xiao P, Gray SJ, Weinberg MS, Samulski RJ (2011) Combination Therapy Utilizing shRNA Knockdown and an Optimized Resistant Transgene for Rescue of Diseases Caused by Mis-folded Proteins. PNAS, 108(34):14258-63. PMID: 21844342.

Gray SJ, Foti SB, Schwartz JW, Bachaboina L, Taylor-Blake B, Coleman J, Ehlers MD, Zylka MJ, McCown TJ, Samulski RJ (2011) Optimizing promoters for rAAV-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Human Gene Therapy, 22(9):1143-53. PMID: 21476867. PMCID: 3177952.

Gray SJ*#, Matagne V*, Bachaboina L, Yadav S, Ojeda S, and Samulski RJ (2011) Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and non-human primates. Molecular Therapy, 19(6):1058-69. PMID: 21487395. PMCID: 3129805. [*equal contributions; #corresponding author]

Snyder BR*, Gray SJ*, Quach ET, Huang JW, Leung CH, Samulski RJ, Boulis NM, Federici T (2011) Comparison of Adeno-Associated Virus Serotypes 1, 6, 8 and 9 for Spinal Cord and Motor Neuron Gene Delivery. Human Gene Therapy, 22(9):1129-35. PMID: 21443428. [*equal contributions]

Dismuke D, Gray SJ, Hirsch M, Samulski RJ, and Muzyczka N (2011) Chapter contribution to Structural Virology, Eds. Agbandje-McKenna and McKenna, RSC, London, UK.

Gray SJ, Woodard KT, Samulski RJ (2010) Viral Vectors and Delivery Strategies for CNS Gene Therapy. Therapeutic Delivery, 1(4):517-534. PMID: 22833965.

Mendell JR, Campbell K, Rodino-Klapac L, Sahenk Z, Shilling C, Lewis S, Bowles D, Gray S, Li C, Galloway G, Malik V, Coley B, Clark KR, Li J, Xiao X, Samulski J, McPhee SW, Samulski RJ, Walker CM. (2010) Dystrophin immunity in Duchenne’s muscular dystrophy. New England Journal of Medicine, 363(15):1429-37. PMID: 20925545. PMCID: 3014106.

Hollis ER 2nd, Jamshidi P, Lorenzana AO, Lee JK, Gray SJ, Samulski RJ, Zheng B, Tuszynski MH (2010) Transient Demyelination Increases the Efficiency of Retrograde AAV Transduction. Molecular Therapy, 18(8):1496-500. PMID: 20502445. PMCID: 2927074.

Gray SJ, Blake B, Criswell HE, Nicolson SC, Samulski RJ, and McCown TJ (2010) Directed Evolution of a Novel Adeno-associated Virus (AAV) Vector that Crosses the Seizure Compromised Blood-Brain Barrier (BBB). Molecular Therapy, 18(3):570-8. PMID: 20040913. PMCID: 2831133.

Hewitt FC, Li C, Gray SJ, Cockrell S, Washburn M, and Samulski RJ (2009) Reducing the Risk of AAV Vector Mobilization with AAV5 Vectors. Journal of Virology, 83(8):3919-29. PMID: 19211760.
Gray SJ andSamulski RJ (2008) Optimizing Gene Delivery Vectors for the Treatment of Heart Disease. Expert Opinion on Biological Therapy, 8(7):911-22. PMID: 18549322.

Gray SJ*, Gerhardt J*, Doerfler W, Small LE, and Fanning E (2007) An Origin of DNA Replication in the Promoter Region of the Human Fragile X Mental Retardation (FMR1) Gene. Molecular and Cellular Biology, 27(2):426-437. PMID: 17101793. [*equal contributions]

Gray SJ, Liu G, Altman AL, Small LE, and Fanning E (2007) Discrete Functional Elements Required for Initiation Activity of the Chinese Hamster Dihydrofolate Reductase Origin Beta at Ectopic Chromosomal Sites. Experimental Cell Research, 313(1):109-20. PMID: 17078947

Daniell H, Datta R, Varma S, Gray SJ, and Lee S (1998) Containment of Herbicide Resistance Through Genetic Manipulation of the Chloroplast Genome. Nature Biotechnology, 16(4):345-348. PMID: 9555724.