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655. Lentiviral Vector Gene Delivery to Mouse CNS by Intrathecal Injections
GENE THERAPY FOR THE NERVOUS SYSTEM II 655. Lentiviral Vector Gene Delivery to Mouse CNS by Intrathecal Injections Elena Fedorova,1 Daniele Marras,1 Ainu Prakash-Cheng,2 G. Luca Gusella.1 1 Medicine, Mount Sinai School of Medicine, New York, NY, United States; 2Human Genetics, Mount Sinai School of Medicine, New York, NY. The possibility of intrathecal gene delivery to the mouse CNS using lentiviral vectors was explored. We used replication-defective, self-inactivating lentiviral vectors carrying the LacZ reporter gene under the control of the cytomegalovirus (CMV) early promoter. Approximately 2x107 transducing units of the lentiviral vector in 20 μl volume were injected into the spinal cord of 2-4-day old C57BL pups. A tracing dye was added to the lentiviral vectors to monitor the success of the injection. The presence of the marker dye in the fontanelles, visible through the skin, indicated the delivery of the lentiviral vector into the spinal cord. One month after a single intrathecal injection, the presence and expression of the LacZ reporter gene was assessed by PCR, in situ PCR, histochemical and immunohistochemical staining. Intrathecal injection of lentiviral vectors containing the LacZ gene produced patchy, widely scattered areas of bacterial β-galactosidase (β-gal) expression. In situ PCR revealed significant levels of the LacZ gene DNA in the cells of the meninges. Positive transduction of the mouse CNS was also confirmed by histochemical and immunohistochemical staining for β-gal. Transgene expression was identified in non-parenchymal cells both on the surface of the spinal cord and on the ventral surface of the brain. No β-gal staining was found in the neural parenchymal elements of the CNS or in nerves. We did not observe histological abnormalities or obvious inflammatory response in the mouse CNS after inoculations. These findings indicate that intrathecal injections of recombinant lentiviral vectors can produce significant levels of transgene expression within the meninges of CNS. Unlike the intracerebral injections of lentiviral vectors, which result in the localized expression of the transgene in the parenchyma near the injection site, intrathecal delivery appears to produce a wider diffusion of the transgene while sparing neural tissue.
656. Retrograde Gene Transduction to Spinal Cord Neurons from Periphery by a ReplicationIncompetent Recombinant HSV-1 Vector Naosuke Nonoguchi,1 James A. Palmer,2 Robert S. Coffin,2 Takuji Watanabe,1 Yoshinaga Kajimoto,1 Hiroto Inoue,1 Toshihiko Kuroiwa,1 Shin-ichi Miyatake.1 1 Neurosurgery, Osaka Medical College, Takatsuki, Osaka, Japan; 2Molecular Pathology, The Windeyer Institute of Medical Sciences, University College London, London, United Kingdom. PURPOSE Herpes Simplex Virus type1 (HSV-1) is suitable as a vector to treat diseases affecting the central nervous system, because of their advantageous properties such as natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. To reduce vector toxicity, many multiple deletion mutants involving various combinations of infected cell proteins (ICP) or viral proteins (VP) have been constructed. We now use the replication-incompetent recombinant HSV-1 strain 1764/4-/pR19LacZ, supplied from a collaborating laboratory of University College London and Biovex Ltd. This vector is disabled by the deletion of ICP4 and ICP34.5 and the inactivation of VP16, and contains the pR19 expression cassette driving the marker gene LacZ under the control of fusion promoter of latency associated transcript promoter and cytomegalovirus enhancer. We performed a Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
study to evaluate the ability of this vector to transduce the gene of interest into CNS neuron including spinal cord. Also the immunogenecity of the vector was investigated. METHOD We used the recombinanat HSV (strain 1764/4-/pR19LacZ) and 8week-old male Wister rats. The 8 μl virus solution (virus titer:1.0x105 pfu/μl) was directly injected into a sciatic nerve by a micro-infuser with a 33 gauge-needle. 15 days after the virus injection, rats were perfused and fixed. Their whole spinal cords and sciatic nerves containing each injection site were removed. All removed organs were stained with X-gal. RESULTS Positive beta-gal activities were detected in the ipsilateral anterior horn motor neurons in spinal cords, with high efficacy, and no inflammatory reaction was seen in all sections. In addition, their leg paralysis was transient in all rats. CONCLUSION We showed that this vector has an ability of efficient retrograde transport to the spinal cord neurons from the periphery and little neuronal toxicity. This recombinant HSV vector is expected to deliver the various genes efficiently to not only spinal cord but also brain. We are preparing the studies of the treatment for the neurodegenerative diseases such as amyotrophic lateral sclerosis and brain ischemia by using this novel HSV-1 vector.
657. Neurotrophic Factor Gene Transfer by AAV Prolongs the Lifespan of Transgenic SOD1Mice Rouman Anguelov,1 Gabor Toth,1 Michael Shy,2 Gyula Acsadi.1,2 Pediatrics, Wayne State University, Detroit, MI; 2Neurology, Wayne State University, Detroit, MI.
1
Preventing or slowing motor neuron degeneration and death in ALS are critical goals of future therapies as are means of enhancing axonal regeneration. A number of neurotrophic factors have been demonstrated to promote survival and/or regeneration of motor neurons both in vitro and in vivo. Previously, we have demonstrated an increase in the lifespan of SOD1 mice, a transgenic animal model for ALS, following injection of a GDNF expressing adenoviral vector in neonatal mice. For human gene therapy, AAV has major advantages over adenoviral vectors. A CMV-GDNF expressing AAV (serotype 2) was constructed using a helper virus free system. The AAV was grown and purified to high titer and injected into hindlimb and paraspinal muscles of 46 or 12 weeks old SOD1 and control mice. The clinical course and motor functions (RotaRod) of mice were followed. SOD1 mice treated with AAV-GDNF after the onset (>12 weeks) of motor symptoms lived longer (136+7.3 days) than SOD1 mice treated at presymptomatic (4-6 weeks) stage (128+8.7) or controls (124+6.5). The onset of hindlimb paralysis was also delayed in the symptomatic group by 9 days. GDNF expression was detected from injected muscles at end-point of experiments. The AAV genome did not reach the spinal cord by retrograde transport. The motor performance (RotaRod) declined less rapidly in the AAV-GDNF injected symptomatic group than in the presymptomatic mice or controls. These data suggest that AAV-mediated GDNF gene transfer in muscles improves the survival and delays the onset of disease when injected into muscles of SOD1 mice even after the onset of motor decline.
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656. Retrograde Gene Transduction to Spinal Cord Neurons from Periphery by a ReplicationIncompetent Recombinant HSV-1 Vector Naosuke Nonoguchi,1 James A. Palmer,2 Robert S. Coffin,2 Takuji Watanabe,1 Yoshinaga Kajimoto,1 Hiroto Inoue,1 Toshihiko Kuroiwa,1 Shin-ichi Miyatake.1 1 Neurosurgery, Osaka Medical College, Takatsuki, Osaka, Japan; 2Molecular Pathology, The Windeyer Institute of Medical Sciences, University College London, London, United Kingdom. PURPOSE Herpes Simplex Virus type1 (HSV-1) is suitable as a vector to treat diseases affecting the central nervous system, because of their advantageous properties such as natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. To reduce vector toxicity, many multiple deletion mutants involving various combinations of infected cell proteins (ICP) or viral proteins (VP) have been constructed. We now use the replication-incompetent recombinant HSV-1 strain 1764/4-/pR19LacZ, supplied from a collaborating laboratory of University College London and Biovex Ltd. This vector is disabled by the deletion of ICP4 and ICP34.5 and the inactivation of VP16, and contains the pR19 expression cassette driving the marker gene LacZ under the control of fusion promoter of latency associated transcript promoter and cytomegalovirus enhancer. We performed a Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
study to evaluate the ability of this vector to transduce the gene of interest into CNS neuron including spinal cord. Also the immunogenecity of the vector was investigated. METHOD We used the recombinanat HSV (strain 1764/4-/pR19LacZ) and 8week-old male Wister rats. The 8 μl virus solution (virus titer:1.0x105 pfu/μl) was directly injected into a sciatic nerve by a micro-infuser with a 33 gauge-needle. 15 days after the virus injection, rats were perfused and fixed. Their whole spinal cords and sciatic nerves containing each injection site were removed. All removed organs were stained with X-gal. RESULTS Positive beta-gal activities were detected in the ipsilateral anterior horn motor neurons in spinal cords, with high efficacy, and no inflammatory reaction was seen in all sections. In addition, their leg paralysis was transient in all rats. CONCLUSION We showed that this vector has an ability of efficient retrograde transport to the spinal cord neurons from the periphery and little neuronal toxicity. This recombinant HSV vector is expected to deliver the various genes efficiently to not only spinal cord but also brain. We are preparing the studies of the treatment for the neurodegenerative diseases such as amyotrophic lateral sclerosis and brain ischemia by using this novel HSV-1 vector.
657. Neurotrophic Factor Gene Transfer by AAV Prolongs the Lifespan of Transgenic SOD1Mice Rouman Anguelov,1 Gabor Toth,1 Michael Shy,2 Gyula Acsadi.1,2 Pediatrics, Wayne State University, Detroit, MI; 2Neurology, Wayne State University, Detroit, MI.
1
Preventing or slowing motor neuron degeneration and death in ALS are critical goals of future therapies as are means of enhancing axonal regeneration. A number of neurotrophic factors have been demonstrated to promote survival and/or regeneration of motor neurons both in vitro and in vivo. Previously, we have demonstrated an increase in the lifespan of SOD1 mice, a transgenic animal model for ALS, following injection of a GDNF expressing adenoviral vector in neonatal mice. For human gene therapy, AAV has major advantages over adenoviral vectors. A CMV-GDNF expressing AAV (serotype 2) was constructed using a helper virus free system. The AAV was grown and purified to high titer and injected into hindlimb and paraspinal muscles of 46 or 12 weeks old SOD1 and control mice. The clinical course and motor functions (RotaRod) of mice were followed. SOD1 mice treated with AAV-GDNF after the onset (>12 weeks) of motor symptoms lived longer (136+7.3 days) than SOD1 mice treated at presymptomatic (4-6 weeks) stage (128+8.7) or controls (124+6.5). The onset of hindlimb paralysis was also delayed in the symptomatic group by 9 days. GDNF expression was detected from injected muscles at end-point of experiments. The AAV genome did not reach the spinal cord by retrograde transport. The motor performance (RotaRod) declined less rapidly in the AAV-GDNF injected symptomatic group than in the presymptomatic mice or controls. These data suggest that AAV-mediated GDNF gene transfer in muscles improves the survival and delays the onset of disease when injected into muscles of SOD1 mice even after the onset of motor decline.
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