| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 20, 2003 as doi:10.1096/fj.03-0567fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Division of Gene Therapy Science,
Department of Otolaryngology and Sensory Organ Surgery, and
Division of Biochemistry, Department of Oncology, Biomedical Research Center B7, Osaka University Graduate School of Medicine, Suita, Japan
2Correspondence: Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: kaneday{at}gts.med.osaka-u.ac.jp
SPECIFIC AIM
No satisfactory therapy for sensorineural hearing impairment is yet available because the auditory sensory epithelium (hair cell (HC)) and its associated neuron (spiral ganglion cell (SGC)) are hardly regenerated in mammalians. We developed a novel gene therapy strategy to prevent and ameliorate hearing impairment by administration of the hemagglutinating virus of Japan envelope (HVJ-E) vector containing hepatocyte growth factor (HGF) gene into the cerebrospinal fluid (CSF).
PRINCIPAL FINDINGS
1. Gene transfer into the inner ear region by intrathecal injection of HVJ-E vector
We developed an effective gene delivery system to the inner ear with minimum invasiveness. We used an HVJ-E vector system, a novel nonviral vector with fusion activity derived from hemagglutinating virus of Japan (HVJ) (Sendai virus). To examine the efficiency, distribution, and safety of the HVJ-E vector, we injected HVJ-E containing marker genes lacZ gene and luciferase gene intrathecally into the CSF of rats via the cisterna magna. No significant damage was observed in either the brain or ear tissues. ß-gal expression was observed in the SGC and stria vascularis, as well as in the cerebral cortex, cerebellum, and medulla. Luciferase activity was detected in the cerebral cortex, medulla, and cochlea from rats injected with the HVJ-E containing luciferase gene but not in other organs such as the lung, spleen, or liver. These results showed that the HVJ-E vector reached the inner ear region after the intrathecal administration and transduced the gene into the tissues without significant damage.
2. In vivo transfection of the HGF gene into the subarachnoid space
We used HGF as a therapeutic molecule for hearing impairment. We administered HVJ-E containing the human HGF gene (hHGF) into the CSF and measured the protein level of HGF by ELISA. Human HGF protein was detected in the CSF of the rats transfected with hHGF even after 12 days of transfection (mean value 0.31 ng/mL on day 5). An increase of rat HGF was also observed in the CSF from the rats administered with hHGF (mean value 2.74 ng/mL on day 5). We immunocytochemically checked exogenous HGF expression in the SGCs obtained from rats inoculated with hHGF (pVAX1-hHGF) and compared the findings with the result from the control group using the control vector (pVAX1) alone. Human HGF was clearly observed in the cytoplasm of SGCs and the percentage of human HGF positive cells was >70%. We next examined the expression of c-Met, a tyrosine kinase receptor of HGF, on SGCs. In rats administered hHGF, the expression level of c-Met was greatly enhanced in SGC cytoplasm.
3. Protective and therapeutic effect of HGF on the inner ear damaged by kanamycin insult
We examined whether HGF can rescue the loss of the HC and SGC induced by kanamycin (KM) treatment. KM was used to mimic the clinical situation of hearing impairment, in which the HC is damaged and lost, leading to the degeneration of SGC due to the lack of neurotrophic substances and electric stimuli. Severe loss of the outer HC and partial loss of the inner HC were observed in the rats inoculated with KM and HVJ-E/pVAX1 (KM+vector group) (Fig. 1
I). However, inner and outer HCs in the rats administered with KM and HVJ-E/pVAX1-hHGF (KM+HGF group), as well as those in the control rat, were well preserved (Fig. 1H, J
). The number of surviving SGCs was assessed. A significant reduction of SGCs was observed in the KM + vector group 4 and 8 wk after KM administration (Fig. 1A
). On the other hand, in the KM + HGF group, the cochlea showed significantly more surviving SGCs on wk 4 and 8 than the KM + vector group. On wk 8, the surviving cell count in the KM + HGF group was 
sixfold higher than those in the KM + vector group (13.3±3.2 cells/10000 µm2 vs. 2.2±1.8 cells/10000 µm2, P<0.05) (Fig. 1A
). These results show that HGF gene transfer has a protective effect on HC and SGC survival. Light microscopic examination demonstrated there were many cells showing vacuolated cytoplasm and nuclei containing clumped chromatin in the KM + vector group (Fig. 1C
). In the KM + HGF group, however, there were considerably fewer cells with such an appearance and most cells had an appearance similar to the control (Fig. 1B, D
). TUNEL staining of SGC showed lower numbers of positive cells in the KM + HGF group than the KM + vector group and control rats (Fig. 1E-G
). These results indicate that the death of HC and SGC in response to KM treatment could be inhibited by the intrathecal HVJ-E inoculation of the HGF gene. Hearing functions before and after KM treatment was also assessed by auditory brainstem response. Hearing impairment was prevented when the HGF gene was administered shortly before KM treatment (Fig. 2
B); even after induction of impairment by KM, hearing function could be recovered (Fig. 2C
).
|
|
CONCLUSIONS AND SIGNIFICANCE
We demonstrated that intrathecal injection of HVJ-E containing hHGF into the CSF prevented the loss of HC and SGC by inhibition of apoptosis and showed a high therapeutic potential for both the prevention and treatment of hearing impairment. The success of this gene therapy is due to two novel issues. One is the novel nonviral vector system; another, the therapeutic molecule with multiple functions.
We used the HVJ-E vector system as a delivery method to the inner ear. HVJ-E vector is constructed by treating HVJ with mild detergent and centrifugation in the presence of plasmid DNA. Our previous studies demonstrated the successful delivery of DNA using this method in vitro and in vivo. In this study, we injected the HVJ-E vector into the CSF to avoid invasion of the inner ear by direct injection to the cochlea. It is thought that HVJ-E most likely spread via the cochlear aqueduct, which connects the CSF to the perilymphatic space of the cochlea. Although safety issues regarding the dissemination of the vector beyond the targeted cochlea need to be addressed, this approach is advantageous, especially for bilateral cochlear gene therapy.
Several neurotrophic factors have been used as therapeutic molecules for the auditory systems. HGF, however, has not been used for this purpose so far. HGF was first identified as a potent mitogen for mature hepatocytes and proved to have multiple functions such as angiogenetic, anti-apoptotic, and neurotrophic activities. These functions of HGF can be enhanced by a positive feedback mechanism, mediated by an essential transcription factor, ETS. In this study, the biological effects of HGF appeared to be up-regulated multifold by such a feedback mechanism, although the level of human HGF in CSF was much lower than rat HGF after stimulation by human HGF. Therefore, HGF gene therapy for the auditory system is thought to have several advantages over the earlier gene therapy using neurotrophic factors. Further study of vascular function in the cochlea after HGF gene transfer will provide novel information regarding cochlear function. Moreover, another possibility exists: this study implies that HGF could cause the regeneration of HC or SGC.
Thus, HGF gene therapy is a potent candidate for treatment of auditory impairment. This research provides new insight and an approach for clinical treatment for hearing impairment by the combination of the HGF gene and the HVJ-E vector system.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0567fje 
This article has been cited by other articles:
|
|
 |
|
  M. Shimamura, N. Sato, S. Waguri, Y. Uchiyama, T. Hayashi, H. Iida, T. Nakamura, T. Ogihara, Y. Kaneda, and R. Morishita Gene Transfer of Hepatocyte Growth Factor Gene Improves Learning and Memory in the Chronic Stage of Cerebral Infarction Hypertension, April 1, 2006; 47(4): 742 - 751. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
