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Retrograde Gene Delivery to Hypoglossal Motoneurons

Permanent Link: http://ufdc.ufl.edu/UFE0045490/00001

Material Information

Title: Retrograde Gene Delivery to Hypoglossal Motoneurons a Possible Therapeutic Approach for Pompe Disease
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Elmallah, Mai Kamal
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: pompe
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Pompe disease is a glycogen storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Patients have macroglossia and tongue motor dysfunction with dysarthria, dysphagia and upper airway obstruction.  A neural basis for tongue dysfunction is indicated by data from the Gaa-/- mouse Pompe model which shows profound glycogen accumulation in hypoglossal motoneurons (XII MNs) and impaired XII motor output. Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools.  Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 following direct tongue injection in 129SVE mice as well as a mouse model which displays neuromuscular pathology (Gaa-/-). Hypoglossal (XII) motoneurons were histologically evaluated 8 wks following tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken beta actin promoter (1x1011 vector genomes (vg)). The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with Gaa-/-. Next, we tested the retrograde transduction of AAV encoding human GAA.  We hypothesized that tongue injection with adeno-associated virus serotype 9 (AAV9) encoding GAA would result in persistent GAA protein expression in XII motoneurons of Gaa-/- mice. AAV9-GAA vector (titer: 1x1011vg; promoter: chicken beta actin, CBA, or Desmin, DES) or lactated ringers solution (sham) was directly injected into the base of the tongue of 2 mo old mice. As expected, GAA could be immunohistochemically detected in XII motoneurons of 129SVE mice but not sham-injected Gaa-/- mice. In contrast, robust GAA immunoreactivity could be detected in Gaa-/- XII motoneurons up to 1 year following AAV9-CBA-GAA tongue injection. Similar results were seen 4 wks following AAV9-DES-GAA injection.  Medullary sections (40 µm) showed that GAA-negative XII motoneurons had a swollen cell body and a granulated appearance. However, Gaa-/- XII MNs that were immunopositive for GAA did not have swollen appearance of the soma. Our data suggest that AAV9 is an effective vector for retrograde gene delivery and gene replacement therapy targeting tongue motor units in Pompe disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mai Kamal Elmallah.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Brantly, Mark Louis.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045490:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045490/00001

Material Information

Title: Retrograde Gene Delivery to Hypoglossal Motoneurons a Possible Therapeutic Approach for Pompe Disease
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Elmallah, Mai Kamal
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: pompe
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Pompe disease is a glycogen storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Patients have macroglossia and tongue motor dysfunction with dysarthria, dysphagia and upper airway obstruction.  A neural basis for tongue dysfunction is indicated by data from the Gaa-/- mouse Pompe model which shows profound glycogen accumulation in hypoglossal motoneurons (XII MNs) and impaired XII motor output. Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools.  Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 following direct tongue injection in 129SVE mice as well as a mouse model which displays neuromuscular pathology (Gaa-/-). Hypoglossal (XII) motoneurons were histologically evaluated 8 wks following tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken beta actin promoter (1x1011 vector genomes (vg)). The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with Gaa-/-. Next, we tested the retrograde transduction of AAV encoding human GAA.  We hypothesized that tongue injection with adeno-associated virus serotype 9 (AAV9) encoding GAA would result in persistent GAA protein expression in XII motoneurons of Gaa-/- mice. AAV9-GAA vector (titer: 1x1011vg; promoter: chicken beta actin, CBA, or Desmin, DES) or lactated ringers solution (sham) was directly injected into the base of the tongue of 2 mo old mice. As expected, GAA could be immunohistochemically detected in XII motoneurons of 129SVE mice but not sham-injected Gaa-/- mice. In contrast, robust GAA immunoreactivity could be detected in Gaa-/- XII motoneurons up to 1 year following AAV9-CBA-GAA tongue injection. Similar results were seen 4 wks following AAV9-DES-GAA injection.  Medullary sections (40 µm) showed that GAA-negative XII motoneurons had a swollen cell body and a granulated appearance. However, Gaa-/- XII MNs that were immunopositive for GAA did not have swollen appearance of the soma. Our data suggest that AAV9 is an effective vector for retrograde gene delivery and gene replacement therapy targeting tongue motor units in Pompe disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mai Kamal Elmallah.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Brantly, Mark Louis.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045490:00001


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1 RETROGRADE GENE DELIVERY TO HYPOGLOSSAL MOTONEURONS: A POSSIBLE THERAPEUTIC APPRO ACH FOR POMPE DISEASE By MAI KAMAL ELMALLAH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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In Vivo Gaa

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Gaa -/Gaa -/

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Gaa -/Gaa -/Gaa -/-

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Gaa -/Gaa -/Gaa -/Gaa -/

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Background and Significance

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Gaa -/

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Tongue and XII Motoneuron Pathology in a Murine Model of Pompe Disease Gaa -/) Gaa -/Gaa Gaa -/Gaa Gaa -/Gaa -/Gaa -/Gaa Gaa -/

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Retrograde AAV Transduction

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Study Aims Aim 1

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Aim 2 Gaa Aim 3 Gaa /

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Gaa -/Gaa -/Gaa -/-

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Gaa -/Gaa -/

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Gaa -/Gaa -/AAV Vectors In Vivo Delivery Gaa

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Gaa -/Gaa -/Gaa -/Vector Pharmacology N en bloc

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Biochemical Assessment

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Brainstem Histology Vibrio cholerae

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Tongue Histology

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Microscopy and Quantitative Analyses Ventilation G aa -/G aa -/N

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Statistical Analysis t

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CT abeling x-none x-none x-none x-nonex-none x-none x-none x-none x-none x-none Retrograde Transport of AAV9 in 129SVE and Gaa -/Mice Evaluation of AAV9-GFP Transduction

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en bloc Gaa -/Gaa -/Evaluation of AAV-GAA Transduction

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Gaa -/Gaa -/Gaa -/Gaa -/Gaa -/en bloc

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Immune Response and Impact on Breathing Weight and ventilation Aims 1 and 2: Weight and ventilation Aim 3: Gaa -/Gaa -/Gaa -/-

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Gaa -/-

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Gaa /

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Gaa -/Gaa -/Gaa -/

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Gaa -/Gaa -/Gaa -/

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Gaa -/Gaa -/Gaa -/-

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Gaa -/Gaa -/Gaa -/

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Commentary on AAV and Retrograde Transport

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Pompe Disease and AAV9

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Conclusion