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Muscle-derived GDNF facilitates motor nerve recovery following sciatic nerve crush injury
TARGETED THERAPIES In utero gene delivery of adenovirus encoded TGFbeta3 restores physiologic palatal fusion and rescues cleft palate in a TGF-beta3 knockout mouse
Muscle-derived GDNF facilitates motor nerve recovery following sciatic nerve crush injury Terence M Myckatyn, MD, Christina Kenney, MD, Alice Tong, MS, Jessica Duan, BS, Daniel Hunter, RA, Alexander Parsadanian, PhD, Susan Mackinnon, MD, FACS Washington University School of Medicine, St Louis, MO
Ryan M Spivak, MS,* Masayuki Endo, MD, PhD, Allison Zajac, BS, Philip Zoltick, MD, Brian Ang, MD, Rebecca Horn, MD, Alan Flake, MD, FACS, Richard Kirschner, MD, Hyun-Duck Nah, DDS, PhD Children’s Hospital of Philadelphia, Philadelphia, PA
INTRODUCTION: GDNF, the most powerful known motor neuron survival factor is also a promising neuroregenerative agent. Evidence suggests that GDNF exerts its neuroregenerative effects in a paracrine manner and so the site of its overexpression will modulate its efficacy.
INTRODUCTION: To date, there has never been a study of fetal gene therapy for a craniofacial disorder. This abstract describes the first such experiment, in which we utilized the TGF-beta3 knockout mouse, which shows complete phenotypic penetration of cleft palate. TGF-beta3, a critical regulator of palatogenesis, is expressed in the medial edge epithelium of the opposing palatal shelves as they fuse on E14.5. Without it, fusion fails, resulting in cleft palate. We propose in utero delivery of virally encoded TGF-beta3 can rescue clefting in the TGF-beta3 knockout mouse.
METHODS: To compare the neuroregenerative effects of GDNF overexpressed either centrally or peripherally, two transgenic murine lines overexpressing GDNF were generated including Myo-GDNF mice (peripheral muscle) and GFAP-GDNF mice (central neuroglia). These mice were bred to thy1-YFP(16) mice whose axons are constitutively labeled with yellow fluorescent protein (YFP). Sciatic nerve crush injuries were evaluated 1, 3, 4, and 6 weeks later using a combination of retrograde labeling, histomorphometry, electron microscopy with non-biased stereology, immunohistochemistry, confocal microscopy of motor endplate reinnervation, and walking track functional analysis.
METHODS: On E11, E12 and E13, fetuses of pregnant TGFbeta3⫹/⫺ females time-mated with TGF-beta3⫹/⫺ males were injected with viral vector under ultrasound. Experimental groups received human adenovirus type-5 vectors encoding GFP and murine TGF-beta3 (Ad-GFP/TGF-beta3). Controls received adenovirus encoded GFP alone (Ad-GFP). On E14.5 and E20, fetuses were harvested, genotyped, and examined by gross and fluorescent stereomicroscopy.
RESULTS: The Myo-GDNF murine line was significantly better than both wild type controls and GFAP-GDNF mice in terms of the density of retrograde labeling of motor neurons, the extent of motor endplate hyperinnervation, and time to functional recovery of walking tracks at all time points tested. Motor endplates were reinnervated more rapidly and with a higher density of terminal axons, and retrograde labeling at 4 weeks following injury showed a profound improvement in motor nerve regeneration compared to all other groups. Modest improvements in nerve regeneration encountered in the GFAP-GDNF mice were not statistically significant.
RESULTS: In the E12 control group, all TGF-beta3 knockout fetuses injected with Ad-GFP demonstrated cleft palate. In the experimental groups, 100% of TGF-beta3 knockouts injected with Ad-GFP/TGF-beta3 exhibited completely fused palates (Table1). Delivery of vector was confirmed by visualization of GFP expression in the palatal shelves and midline. In addition, TGFbeta3 knockouts injected with Ad-GFP/TGF-beta3 and harvested on E14.5 demonstrated anterior to posterior fusion of the palatal shelves.
Time of Injection
Vector injected
# TGFbeta3 ⴙ/ⴙ fetuses injected
# TGFbeta3 ⴙ/ⴚ fetuses injected
# TGFbeta3 ⴚ/ⴚ fetuses injected
Total # fetuses injected
% TGFbeta3 ⴚ/ⴚ fetuses with complete rescue
E11
Ad-GFP/ TGFbeta3
3
6
3
12
100%
E12
Ad-GFP/ TGFbeta3
13
16
6
35
100%
E12
Ad-GFP
3
7
2
12
-
E13
Ad-GFP/ TGFbeta3
4
7
3
14
100%
CONCLUSIONS: Our findings support the development of strategies to optimize the delivery of GDNF into peripheral muscle rather than into the central nervous system to accelerate motor recovery following nerve injury.
Eukaryotic Initiation Factor 4E (eIF4E) can be effectively downregulated using small interfering RNA (siRNA), inhibiting growth in breast cancer cells Alpana Soni, MD, Argun Akcakanat, MD, PhD, Funda Meric-Bernstam, MD University of Texas, MD Anderson Cancer Center, Houston, TX INTRODUCTION: Dysregulation of translation is critical to malignant cell growth. Eukaryotic initiation factor 4E (eIF4E) is the ratelimiting factor in cap-dependent translation, and its bioavailability is strictly controlled through its association with eIF4E-binding protein 1 (4E-BP1). eIF4E is overexpressed in several cancer types including breast cancer, and has been linked to a poor prognosis. We sought to determine whether eIF4E-knockdown would decrease breast cancer cell growth.
CONCLUSIONS: In utero delivery of virally encoded TGF-beta3 restores the physiologic fusion process of the palate and rescues clefting in the TGF-beta3 knockout mouse.
© 2007 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/07/$32.00
S92
Muscle-derived GDNF facilitates motor nerve recovery following sciatic nerve crush injury Terence M Myckatyn, MD, Christina Kenney, MD, Alice Tong, MS, Jessica Duan, BS, Daniel Hunter, RA, Alexander Parsadanian, PhD, Susan Mackinnon, MD, FACS Washington University School of Medicine, St Louis, MO
Ryan M Spivak, MS,* Masayuki Endo, MD, PhD, Allison Zajac, BS, Philip Zoltick, MD, Brian Ang, MD, Rebecca Horn, MD, Alan Flake, MD, FACS, Richard Kirschner, MD, Hyun-Duck Nah, DDS, PhD Children’s Hospital of Philadelphia, Philadelphia, PA
INTRODUCTION: GDNF, the most powerful known motor neuron survival factor is also a promising neuroregenerative agent. Evidence suggests that GDNF exerts its neuroregenerative effects in a paracrine manner and so the site of its overexpression will modulate its efficacy.
INTRODUCTION: To date, there has never been a study of fetal gene therapy for a craniofacial disorder. This abstract describes the first such experiment, in which we utilized the TGF-beta3 knockout mouse, which shows complete phenotypic penetration of cleft palate. TGF-beta3, a critical regulator of palatogenesis, is expressed in the medial edge epithelium of the opposing palatal shelves as they fuse on E14.5. Without it, fusion fails, resulting in cleft palate. We propose in utero delivery of virally encoded TGF-beta3 can rescue clefting in the TGF-beta3 knockout mouse.
METHODS: To compare the neuroregenerative effects of GDNF overexpressed either centrally or peripherally, two transgenic murine lines overexpressing GDNF were generated including Myo-GDNF mice (peripheral muscle) and GFAP-GDNF mice (central neuroglia). These mice were bred to thy1-YFP(16) mice whose axons are constitutively labeled with yellow fluorescent protein (YFP). Sciatic nerve crush injuries were evaluated 1, 3, 4, and 6 weeks later using a combination of retrograde labeling, histomorphometry, electron microscopy with non-biased stereology, immunohistochemistry, confocal microscopy of motor endplate reinnervation, and walking track functional analysis.
METHODS: On E11, E12 and E13, fetuses of pregnant TGFbeta3⫹/⫺ females time-mated with TGF-beta3⫹/⫺ males were injected with viral vector under ultrasound. Experimental groups received human adenovirus type-5 vectors encoding GFP and murine TGF-beta3 (Ad-GFP/TGF-beta3). Controls received adenovirus encoded GFP alone (Ad-GFP). On E14.5 and E20, fetuses were harvested, genotyped, and examined by gross and fluorescent stereomicroscopy.
RESULTS: The Myo-GDNF murine line was significantly better than both wild type controls and GFAP-GDNF mice in terms of the density of retrograde labeling of motor neurons, the extent of motor endplate hyperinnervation, and time to functional recovery of walking tracks at all time points tested. Motor endplates were reinnervated more rapidly and with a higher density of terminal axons, and retrograde labeling at 4 weeks following injury showed a profound improvement in motor nerve regeneration compared to all other groups. Modest improvements in nerve regeneration encountered in the GFAP-GDNF mice were not statistically significant.
RESULTS: In the E12 control group, all TGF-beta3 knockout fetuses injected with Ad-GFP demonstrated cleft palate. In the experimental groups, 100% of TGF-beta3 knockouts injected with Ad-GFP/TGF-beta3 exhibited completely fused palates (Table1). Delivery of vector was confirmed by visualization of GFP expression in the palatal shelves and midline. In addition, TGFbeta3 knockouts injected with Ad-GFP/TGF-beta3 and harvested on E14.5 demonstrated anterior to posterior fusion of the palatal shelves.
Time of Injection
Vector injected
# TGFbeta3 ⴙ/ⴙ fetuses injected
# TGFbeta3 ⴙ/ⴚ fetuses injected
# TGFbeta3 ⴚ/ⴚ fetuses injected
Total # fetuses injected
% TGFbeta3 ⴚ/ⴚ fetuses with complete rescue
E11
Ad-GFP/ TGFbeta3
3
6
3
12
100%
E12
Ad-GFP/ TGFbeta3
13
16
6
35
100%
E12
Ad-GFP
3
7
2
12
-
E13
Ad-GFP/ TGFbeta3
4
7
3
14
100%
CONCLUSIONS: Our findings support the development of strategies to optimize the delivery of GDNF into peripheral muscle rather than into the central nervous system to accelerate motor recovery following nerve injury.
Eukaryotic Initiation Factor 4E (eIF4E) can be effectively downregulated using small interfering RNA (siRNA), inhibiting growth in breast cancer cells Alpana Soni, MD, Argun Akcakanat, MD, PhD, Funda Meric-Bernstam, MD University of Texas, MD Anderson Cancer Center, Houston, TX INTRODUCTION: Dysregulation of translation is critical to malignant cell growth. Eukaryotic initiation factor 4E (eIF4E) is the ratelimiting factor in cap-dependent translation, and its bioavailability is strictly controlled through its association with eIF4E-binding protein 1 (4E-BP1). eIF4E is overexpressed in several cancer types including breast cancer, and has been linked to a poor prognosis. We sought to determine whether eIF4E-knockdown would decrease breast cancer cell growth.
CONCLUSIONS: In utero delivery of virally encoded TGF-beta3 restores the physiologic fusion process of the palate and rescues clefting in the TGF-beta3 knockout mouse.
© 2007 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/07/$32.00
S92