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394. Brain Penetrating Gene Vectors for Efficient Gene Transfer To the CNS
NEUROLOGICAL DISEASES I ScFv antibodies against the RRM1 domain of TDP-43 will form the basis for the novel antibody-based therapy that specifically targets TDP-43-induced inflammation and proteinopathy. Moreover, it will constitute a new and useful tool to better elucidate the mechanisms of TDP-43 toxicity. References (1) Neumann M, et al. Science 2006. (2) Swarup V, et al. JEM 2011. (3) Chang C, et al. FEBS Letters 2013. (4) Shodai A, et al. JBC 2013. (5) Patel P, et al. Mol. Ther. 2013.
393. Endogenous Gene Activation in the Brain of an Angelman Syndrome Mouse Model Barbara J. Bailus,1 Benjamin R. Pyles,1 David J. Segal.1 1 Genome Center and Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA.
We have developed a therapeutic protein that can be injected into the peritoneum of a mouse, cross the blood brain barrier, and modulate expression of a gene in neurons throughout the brain. The protein is a zinc finger-based artificial transcription factor designed to activate expression of Ube3a, the loss of which in humans causes Angelman Syndrome. Epigenetic imprinting causes normal Ube3a expression from only the maternal allele, which is typically lost due to deletion in Angelman Syndrome. The paternal allele is intact but epigenetically silenced by a long anti-sense transcript. The therapeutic protein silences the Ube3a antisense transcript, enabling endogenous expression of the paternal Ube3a. Live animal imaging demonstrated a peak concentration in brain at four hours post injection. Immunofluorescence and western blot demonstrate Ube3a activation, with good distribution compared to viral vectors. We will also discuss behavioral response to treatment, and compare immune response to other platforms such as TALE and CRISPR proteins. If generalizable, this methodology could be useful for the treatment of many brain disorders.
394. Brain Penetrating Gene Vectors for Efficient Gene Transfer To the CNS
Panagiotis Mastorakos,1 Clark Zhang,1 Sneha Berry,1 Anthony Kim,2 Graeme Woodworth,2 Jung Soo Suk,1 Justin Hanes.1 1 Johns Hopkins University School of Medicine, Baltimore, MD; 2 Neurological Surgery, University of Maryland, Baltimore, MD. Introduction Gene therapy constitutes an attractive approach for curing a variety of CNS diseases. Viral gene delivery, though relatively efficient, has been limited by immunogenicity, low packaging capacity, difficulties in scale-up. Non-viral gene vectors offer an alternate strategy but face a number of obstacles to reach the target cells. While acknowledging the well- characterized blood brain barrier as a primary limitation, we focus our studies on surpassing the anisotropic and electrostatically charged extracellular space (ECS) found between brain cells. This ‘brain tissue barrier’, regardless of administration method, hampers widespread distribution of gene vectors in the brain, thereby limiting the gene transfer to target cells. Previous studies have shown that exceptionally well coated sub-100 nm nanoparticles can rapidly diffuse in the brain parenchyma allowing the widespread distribution of therapeutics [1]. We developed a polyethyleneimine (PEI) based, small and stable nanoparticle with a sufficient PEG coating to rapidly penetrate the brain parenchyma. Methods Densely PEGylated PEI nanoparticles were formulated as previously described [2]. Physicochemical properties were determined in 10 mM NaCl and artificial CSF using a Zetasizer NanoZS. Multiple particle tracking (MPT) was used to estimate the mean square displacement of fluorescent gene vectors in ex vivo rodent brain slices S150
[1]. We assessed in vivo distribution and transfection of gene vectors using Zeiss LSM 710 confocal microscope following convection enhanced delivery (CED) to the rat striatum. Results and Discussion We formulated near neutral 50 nm brain penetrating nanoparticles (BPN) and compared them to conventionally PEGylated (CPN) and un-PEGylated (UPN) PEI nanoparticles. Dense PEGylation dramatically increases stability of the gene vectors and decreases in vitro toxicity. Based on our MPT analysis, BPNs rapidly diffuse in brain ECM whereas CPNs and UPNs appear to be significantly restricted in their motion. Following CED, fluorescently labeled BPNs covered a ~3 fold higher volume of the striatum than labeled CPNs and UPNs. Accordingly, the administration of unlabeled BPNs carrying eGFP encoding plasmids led to a ~3 fold increase in the volume in which cells were reached and transfected compared to unlabeled CPN carrying the same plasmids. Our results indicate that a dense PEG coating can allow an efficient penetration and distribution of gene vectors in the brain parenchyma thus providing a widespread gene transfer to the brain tissues. We believe that this novel strategy of brain penetrating gene vector formulation can be applied to a variety of cationic polymers that have been designed to overcome other barriers, further enhancing gene transfer to the brain. 1. Nance, E.A., et al., A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue. Sci Transl Med, 2012. 4(149): p. 149ra119. 2. Suk, J., Kim, A., et al., Lung Gene Therapy with Highly Compacted DNA Nanoparticles that Overcome the Mucus Barrier. Journal of Controlled Release, In Press.
395. OXB-102: An Enhanced Gene Therapy for Parkinson’s Disease
Romina A. Badin,1 Katie M. Binley,2 Nadja VanCamp,1 Caroline Jan,1 Jeanne Gourlay,1 Hannah Stewart,2 Scott Ralph,2 Yatish Lad,2 Koichi Hosomi,1,3 Stephane Palfi,1,3 Phillippe Hantraye,1 Kyriacos Mitrophanous.2 1 CEA CNRS 2210 MIRCEN, Fontenay-aux-Roses, Paris, France; 2Oxford BioMedica (UK) Ltd, Oxford, United Kingdom; 3 Neurosurgery, Henri Mondor Hospital, Creteil, France. Oral dopaminergic treatments are the primary standard of care for Parkinson’s disease (PD); although these are highly efficacious early on, over time they lead to debilitating long term side effects that seriously impact on quality of life and restrict the longevity of such treatments. OXB-102 is a lentiviral vector derived from the equine infectious anaemia virus (EIAV) that delivers the genes encoding the three key enzymes in the dopamine (DA) biosynthetic pathway, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP-cyclohydrolase (CH1), to non-dopaminergic striatal neurons of the sensorimotor putamen, thus providing these cells with the ability to synthesize and release their own DA. The effectiveness of this strategy has already been demonstrated in rodents, non-human primates and Parkinson’s (PD) patients with a precursor gene therapy vector called ProSavin®. OXB-102 is an improved version of ProSavin® that expresses the same enzymes but with increased DA production. In vitro evaluation in human primary neuronal cultures showed that OXB-102 is at least 7 fold more potent in terms of DA production than an equivalent dose of ProSavin®. In non-clinical studies the efficacy of a full-strength (FD) and a 1/5th (LD) dose of OXB-102 has been compared to the efficacy of full-strength ProSavin® in the ‘gold standard’ MPTP macaque model of PD; a vector expressing the LacZ reporter gene (LacZ) was used as a negative control. The longitudinal follow-up consisted of recording Clinical Rating Scores (CRS) and video-based quantification of locomotor activity before and after vector injection. The full-strength and the 1/5th doses of Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
393. Endogenous Gene Activation in the Brain of an Angelman Syndrome Mouse Model Barbara J. Bailus,1 Benjamin R. Pyles,1 David J. Segal.1 1 Genome Center and Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA.
We have developed a therapeutic protein that can be injected into the peritoneum of a mouse, cross the blood brain barrier, and modulate expression of a gene in neurons throughout the brain. The protein is a zinc finger-based artificial transcription factor designed to activate expression of Ube3a, the loss of which in humans causes Angelman Syndrome. Epigenetic imprinting causes normal Ube3a expression from only the maternal allele, which is typically lost due to deletion in Angelman Syndrome. The paternal allele is intact but epigenetically silenced by a long anti-sense transcript. The therapeutic protein silences the Ube3a antisense transcript, enabling endogenous expression of the paternal Ube3a. Live animal imaging demonstrated a peak concentration in brain at four hours post injection. Immunofluorescence and western blot demonstrate Ube3a activation, with good distribution compared to viral vectors. We will also discuss behavioral response to treatment, and compare immune response to other platforms such as TALE and CRISPR proteins. If generalizable, this methodology could be useful for the treatment of many brain disorders.
394. Brain Penetrating Gene Vectors for Efficient Gene Transfer To the CNS
Panagiotis Mastorakos,1 Clark Zhang,1 Sneha Berry,1 Anthony Kim,2 Graeme Woodworth,2 Jung Soo Suk,1 Justin Hanes.1 1 Johns Hopkins University School of Medicine, Baltimore, MD; 2 Neurological Surgery, University of Maryland, Baltimore, MD. Introduction Gene therapy constitutes an attractive approach for curing a variety of CNS diseases. Viral gene delivery, though relatively efficient, has been limited by immunogenicity, low packaging capacity, difficulties in scale-up. Non-viral gene vectors offer an alternate strategy but face a number of obstacles to reach the target cells. While acknowledging the well- characterized blood brain barrier as a primary limitation, we focus our studies on surpassing the anisotropic and electrostatically charged extracellular space (ECS) found between brain cells. This ‘brain tissue barrier’, regardless of administration method, hampers widespread distribution of gene vectors in the brain, thereby limiting the gene transfer to target cells. Previous studies have shown that exceptionally well coated sub-100 nm nanoparticles can rapidly diffuse in the brain parenchyma allowing the widespread distribution of therapeutics [1]. We developed a polyethyleneimine (PEI) based, small and stable nanoparticle with a sufficient PEG coating to rapidly penetrate the brain parenchyma. Methods Densely PEGylated PEI nanoparticles were formulated as previously described [2]. Physicochemical properties were determined in 10 mM NaCl and artificial CSF using a Zetasizer NanoZS. Multiple particle tracking (MPT) was used to estimate the mean square displacement of fluorescent gene vectors in ex vivo rodent brain slices S150
[1]. We assessed in vivo distribution and transfection of gene vectors using Zeiss LSM 710 confocal microscope following convection enhanced delivery (CED) to the rat striatum. Results and Discussion We formulated near neutral 50 nm brain penetrating nanoparticles (BPN) and compared them to conventionally PEGylated (CPN) and un-PEGylated (UPN) PEI nanoparticles. Dense PEGylation dramatically increases stability of the gene vectors and decreases in vitro toxicity. Based on our MPT analysis, BPNs rapidly diffuse in brain ECM whereas CPNs and UPNs appear to be significantly restricted in their motion. Following CED, fluorescently labeled BPNs covered a ~3 fold higher volume of the striatum than labeled CPNs and UPNs. Accordingly, the administration of unlabeled BPNs carrying eGFP encoding plasmids led to a ~3 fold increase in the volume in which cells were reached and transfected compared to unlabeled CPN carrying the same plasmids. Our results indicate that a dense PEG coating can allow an efficient penetration and distribution of gene vectors in the brain parenchyma thus providing a widespread gene transfer to the brain tissues. We believe that this novel strategy of brain penetrating gene vector formulation can be applied to a variety of cationic polymers that have been designed to overcome other barriers, further enhancing gene transfer to the brain. 1. Nance, E.A., et al., A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue. Sci Transl Med, 2012. 4(149): p. 149ra119. 2. Suk, J., Kim, A., et al., Lung Gene Therapy with Highly Compacted DNA Nanoparticles that Overcome the Mucus Barrier. Journal of Controlled Release, In Press.
395. OXB-102: An Enhanced Gene Therapy for Parkinson’s Disease
Romina A. Badin,1 Katie M. Binley,2 Nadja VanCamp,1 Caroline Jan,1 Jeanne Gourlay,1 Hannah Stewart,2 Scott Ralph,2 Yatish Lad,2 Koichi Hosomi,1,3 Stephane Palfi,1,3 Phillippe Hantraye,1 Kyriacos Mitrophanous.2 1 CEA CNRS 2210 MIRCEN, Fontenay-aux-Roses, Paris, France; 2Oxford BioMedica (UK) Ltd, Oxford, United Kingdom; 3 Neurosurgery, Henri Mondor Hospital, Creteil, France. Oral dopaminergic treatments are the primary standard of care for Parkinson’s disease (PD); although these are highly efficacious early on, over time they lead to debilitating long term side effects that seriously impact on quality of life and restrict the longevity of such treatments. OXB-102 is a lentiviral vector derived from the equine infectious anaemia virus (EIAV) that delivers the genes encoding the three key enzymes in the dopamine (DA) biosynthetic pathway, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP-cyclohydrolase (CH1), to non-dopaminergic striatal neurons of the sensorimotor putamen, thus providing these cells with the ability to synthesize and release their own DA. The effectiveness of this strategy has already been demonstrated in rodents, non-human primates and Parkinson’s (PD) patients with a precursor gene therapy vector called ProSavin®. OXB-102 is an improved version of ProSavin® that expresses the same enzymes but with increased DA production. In vitro evaluation in human primary neuronal cultures showed that OXB-102 is at least 7 fold more potent in terms of DA production than an equivalent dose of ProSavin®. In non-clinical studies the efficacy of a full-strength (FD) and a 1/5th (LD) dose of OXB-102 has been compared to the efficacy of full-strength ProSavin® in the ‘gold standard’ MPTP macaque model of PD; a vector expressing the LacZ reporter gene (LacZ) was used as a negative control. The longitudinal follow-up consisted of recording Clinical Rating Scores (CRS) and video-based quantification of locomotor activity before and after vector injection. The full-strength and the 1/5th doses of Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy