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Risk assessments in infants suspect having later-onset Pompe disease identified through newborn screening
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Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
56 Risk assessments in infants suspect having later-onset Pompe disease identified through newborn screening Yin-Hsiu Chien, Pin-Wen Chen, Ping-Chun Wang, Shu-Chuan Chiang, Wuh-Liang Hwu, National Taiwan University Hospital, Taipei, Taiwan Purpose: Newborn screening (NBS) for Pompe disease detects infants with both infantile-onset and later-onset Pompe disease. A two-tier strategy combining acid alpha-glucosidase activity measurement and cardiac evaluation can easily diagnose infants with cardiac involvement and initiate the treatment immediately. However, for infants without cardiac involvement, suspecting later-onset Pompe disease (LOPD), it is crucial to predict their risks to develop symptoms during childhood. Methods: We determined urinary glucose tetrasaccharide (Glc4) concentrations, a marker of glycogen storage and tissue damage, acid alpha-glucosidase in lymphocytes, and serum creatine kinase (CK) levels, and genotypes in infants suspect having LOPD (n =29) identified through newborn screening from 2005 to 2015. Comparison groups included 1) classic infantile-onset Pompe disease (n=12) defined as patients with evidences for hypertrophic cardiomyopathy by EKG, X-ray and echocardiogram, and 2) false positives infants (n=33). Urinary Glc4 concentrations were determined using tandem mass spectrometry. Results: Baseline Glc4 concentrations were at or above the 90th percentile of the age-matched reference range in all IOPD patients. All of them also showed marked elevation of CK levels at baseline. 14 out of the total 29 LOPD infants had elevation of baseline Glc4 concentrations, but 6 having normal serum CK at baseline. The high risk group, defining as elevation of CK and Glc4, contained 12 IOPD patients and 8 LOPD infants. Five of the 8 LOPD high risk infants were already on enzyme replacement therapy (ERT). Thirteen infants with normal CK and normal Glc4 were classified as low-risk group, and most of their genotypes were novel pathologic mutations. Conclusion: The combination risk assessment correlated well with the genotypes, and probably can serve as a useful reference for infants suspect having LOPD identified through NBS.
doi:10.1016/j.ymgme.2016.11.065
57 Dose-ranging comparison of choroid plexus-directed versus pan-neuronal-directed recombinant AAV gene therapy in a murine model of alpha-mannosidosis Eun-Young Choi, Stephen G Kaler, NIH, Bethesda, MD, United States In a dose-ranging study of recombinant adeno-associated viral (rAAV) serotypes of variable tropism in a mouse model of alphamannosidosis, we compared the respective biochemical and pathological effects, and safety profiles. On administration to the cerebrospinal fluid (CSF) of mice, rAAV serotype 5 selectively transduces choroid plexus epithelia. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time CSF administration of a rAAV could be an attractive strategy for long-term treatment of lysosomal storage diseases (LSDs). Here, we compare the biochemical effects of choroid plexus-directed rAAV5 gene therapy for lysosomal acid-alpha-mannosidase (LAMAN) deficiency to that obtained with rAAV9, rAAV4, and rAAV6. We cloned the human (hu) LAMAN cDNA into a rAAV shuttle plasmid and generated rAAV4, rAAV5, rAAV6 and rAAV9 expressing huLAMAN. We administered viral particles to the CSF
of homozygous mutant mice by brain lateral ventricle injection on day 3 of life at doses of 5 x 109, 1.6 x 1010, or 5 x 1010. We documented dose-dependent transduction and huLAMAN mRNA expression in the choroid plexuses of rAAV-treated animals. In addition to choroid plexus, rAAV9-treated animals showed pan-neuronal transduction, resulting in higher overall LAMAN enzyme activity throughout the brain. No significant clinical or pathological improvement was associated with the increased LAMAN activity in this mouse model, however. In a concurrent natural history study of alpha-mannosidosis at NIH, we noted distinctive abnormalities on brain magnetic resonance spectroscopy, and magnetic resonance imaging of lumbar spine in four adult patients. Proteomics evaluation of CSF collected under protocol from these subjects is in progress, to establish a CSF biomarker for potential use in a future first-in-human viral gene therapy trial for this illness. The optimal approach rAAV gene therapy for alpha-mannosidosis remains debatable; the overarching goal of this study is to clarify the safest and most effective elements.
doi:10.1016/j.ymgme.2016.11.066
58 Delivery of lysosomal enzymes via EXPLOR: exosome engineered for protein loading by optogenetically reversible protein interaction Hojun Choia, Nambin Yima, Seung-Wook Ryub, Kyungsun Choia, Chulhee Choia,b, aKAIST, Daejeon, Republic of Korea, bCellex Life Sciences, Incorporated, Daejeon, Republic of Korea Many intracellular proteins with great potential as biopharmaceutical drugs have been identified; however, many of the challenges associated with intracellular protein delivery have yet to be solved. Although protein transduction and lipid nanoparticle-mediated protein delivery methods have been proposed for direct protein delivery into target cells and tissues, many obstacles remain before these methods can be successfully employed in vivo, including low purification efficiency, failure to separate from nanoparticles in recipient cells, and induction of immune responses against host immune cells. To address these limitations, we developed an optogenetically engineered exosome system, named "exosomes for protein loading via optically reversible protein-protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein-protein interactions (PPI). Among nanoparticles, cell-derived exosomes have recently been highlighted as new therapeutic strategies for the in vivo delivery of nucleotides and chemical drugs. Recent studies have sought to use exosomes as a new method for the in vivo delivery of siRNA or miRNA to specific target tissues by systemic injection. These methods were based on the passive loading of siRNAs or miRNAs into isolated exosomes by electrophoresis, a method poorly suited for the intracellular delivery of cellular proteins. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, we were able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLOR was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dosedependent manner. In the present study, we have demonstrated the intracellular delivery of beta-glucocerebrosidase as functional proteins in the target cells and in vivo. These results clearly indicate the potential of EXPLOR for treatment of Gaucher disease.
doi:10.1016/j.ymgme.2016.11.067
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
56 Risk assessments in infants suspect having later-onset Pompe disease identified through newborn screening Yin-Hsiu Chien, Pin-Wen Chen, Ping-Chun Wang, Shu-Chuan Chiang, Wuh-Liang Hwu, National Taiwan University Hospital, Taipei, Taiwan Purpose: Newborn screening (NBS) for Pompe disease detects infants with both infantile-onset and later-onset Pompe disease. A two-tier strategy combining acid alpha-glucosidase activity measurement and cardiac evaluation can easily diagnose infants with cardiac involvement and initiate the treatment immediately. However, for infants without cardiac involvement, suspecting later-onset Pompe disease (LOPD), it is crucial to predict their risks to develop symptoms during childhood. Methods: We determined urinary glucose tetrasaccharide (Glc4) concentrations, a marker of glycogen storage and tissue damage, acid alpha-glucosidase in lymphocytes, and serum creatine kinase (CK) levels, and genotypes in infants suspect having LOPD (n =29) identified through newborn screening from 2005 to 2015. Comparison groups included 1) classic infantile-onset Pompe disease (n=12) defined as patients with evidences for hypertrophic cardiomyopathy by EKG, X-ray and echocardiogram, and 2) false positives infants (n=33). Urinary Glc4 concentrations were determined using tandem mass spectrometry. Results: Baseline Glc4 concentrations were at or above the 90th percentile of the age-matched reference range in all IOPD patients. All of them also showed marked elevation of CK levels at baseline. 14 out of the total 29 LOPD infants had elevation of baseline Glc4 concentrations, but 6 having normal serum CK at baseline. The high risk group, defining as elevation of CK and Glc4, contained 12 IOPD patients and 8 LOPD infants. Five of the 8 LOPD high risk infants were already on enzyme replacement therapy (ERT). Thirteen infants with normal CK and normal Glc4 were classified as low-risk group, and most of their genotypes were novel pathologic mutations. Conclusion: The combination risk assessment correlated well with the genotypes, and probably can serve as a useful reference for infants suspect having LOPD identified through NBS.
doi:10.1016/j.ymgme.2016.11.065
57 Dose-ranging comparison of choroid plexus-directed versus pan-neuronal-directed recombinant AAV gene therapy in a murine model of alpha-mannosidosis Eun-Young Choi, Stephen G Kaler, NIH, Bethesda, MD, United States In a dose-ranging study of recombinant adeno-associated viral (rAAV) serotypes of variable tropism in a mouse model of alphamannosidosis, we compared the respective biochemical and pathological effects, and safety profiles. On administration to the cerebrospinal fluid (CSF) of mice, rAAV serotype 5 selectively transduces choroid plexus epithelia. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time CSF administration of a rAAV could be an attractive strategy for long-term treatment of lysosomal storage diseases (LSDs). Here, we compare the biochemical effects of choroid plexus-directed rAAV5 gene therapy for lysosomal acid-alpha-mannosidase (LAMAN) deficiency to that obtained with rAAV9, rAAV4, and rAAV6. We cloned the human (hu) LAMAN cDNA into a rAAV shuttle plasmid and generated rAAV4, rAAV5, rAAV6 and rAAV9 expressing huLAMAN. We administered viral particles to the CSF
of homozygous mutant mice by brain lateral ventricle injection on day 3 of life at doses of 5 x 109, 1.6 x 1010, or 5 x 1010. We documented dose-dependent transduction and huLAMAN mRNA expression in the choroid plexuses of rAAV-treated animals. In addition to choroid plexus, rAAV9-treated animals showed pan-neuronal transduction, resulting in higher overall LAMAN enzyme activity throughout the brain. No significant clinical or pathological improvement was associated with the increased LAMAN activity in this mouse model, however. In a concurrent natural history study of alpha-mannosidosis at NIH, we noted distinctive abnormalities on brain magnetic resonance spectroscopy, and magnetic resonance imaging of lumbar spine in four adult patients. Proteomics evaluation of CSF collected under protocol from these subjects is in progress, to establish a CSF biomarker for potential use in a future first-in-human viral gene therapy trial for this illness. The optimal approach rAAV gene therapy for alpha-mannosidosis remains debatable; the overarching goal of this study is to clarify the safest and most effective elements.
doi:10.1016/j.ymgme.2016.11.066
58 Delivery of lysosomal enzymes via EXPLOR: exosome engineered for protein loading by optogenetically reversible protein interaction Hojun Choia, Nambin Yima, Seung-Wook Ryub, Kyungsun Choia, Chulhee Choia,b, aKAIST, Daejeon, Republic of Korea, bCellex Life Sciences, Incorporated, Daejeon, Republic of Korea Many intracellular proteins with great potential as biopharmaceutical drugs have been identified; however, many of the challenges associated with intracellular protein delivery have yet to be solved. Although protein transduction and lipid nanoparticle-mediated protein delivery methods have been proposed for direct protein delivery into target cells and tissues, many obstacles remain before these methods can be successfully employed in vivo, including low purification efficiency, failure to separate from nanoparticles in recipient cells, and induction of immune responses against host immune cells. To address these limitations, we developed an optogenetically engineered exosome system, named "exosomes for protein loading via optically reversible protein-protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein-protein interactions (PPI). Among nanoparticles, cell-derived exosomes have recently been highlighted as new therapeutic strategies for the in vivo delivery of nucleotides and chemical drugs. Recent studies have sought to use exosomes as a new method for the in vivo delivery of siRNA or miRNA to specific target tissues by systemic injection. These methods were based on the passive loading of siRNAs or miRNAs into isolated exosomes by electrophoresis, a method poorly suited for the intracellular delivery of cellular proteins. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, we were able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLOR was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dosedependent manner. In the present study, we have demonstrated the intracellular delivery of beta-glucocerebrosidase as functional proteins in the target cells and in vivo. These results clearly indicate the potential of EXPLOR for treatment of Gaucher disease.
doi:10.1016/j.ymgme.2016.11.067