Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles.
BACKGROUND
Direct gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1) particles that contain chimeric glycoprotein C (gC) - glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) proteins. HSV-1 vector particles containing either gC - GDNF or gC - BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH) promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression.


RESULTS
Helper virus-free HSV-1 vector packaging was performed using either gC - GDNF or gC - BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal-specific promoters improved nigrostriatal neuron-specific expression (83 to 93%) compared to either approach alone, and supported long-term (1 month) expression at levels similar to those observed using untargeted gene transfer.


CONCLUSIONS
Targeted gene transfer can be used in combination with neuronal-specific promoters to achieve a high level of nigrostriatal neuron-specific expression. Targeted gene transfer can be followed by long-term expression. Nigrostriatal neuron-specific expression may be useful for specific gene therapy approaches to Parkinson's disease or for genetic analyses of nigrostriatal neuron physiology.
Cao
Haiyan
H
Department of Neurology, West Roxbury VA Hospital/Harvard Medical School, W, Roxbury, MA 02132, USA. haiyan_cao@hms.harvard.edu
Zhang
Guo-rong
GR
Wang
Xiaodan
X
Kong
Lingxin
L
Geller
Alfred I
AI
D000818:Animals; D001370:Axonal Transport / Q000235:genetics; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003342:Corpus Striatum / Q000378:metabolism*; D006224:Cricetinae; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008297:Male; D009434:Neural Pathways / Q000378:metabolism; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013378:Substantia Nigra / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D051100:Glial Cell Line-Derived Neurotrophic Factor; D016900:Neurofilament Proteins; D011993:Recombinant Fusion Proteins; C051774:neurofilament protein H; D014446:Tyrosine 3-Monooxygenase