The Study On Construction Of Human Foamy Virus Vector And Its Potential Application For The Treatment Of Neurological Diseases

Human foamy virus (HFV), the first identified human retrovirus, was isolated in 1971 from a patient with a nasopharyngeal carcinoma. Similar to other retroviruses, such as human immunodeficiency virus (HIV) and human T cell leukemia virus, the HFV genome encodes the canonical retroviral gag, pol, and env genes, as well as several additional genes termed bel-1 (also known as tas), bel-2 and bel-3. Interestingly, it was found that HFV is unique in that, in addition to a promoter in the U3 (3'unique) region of the long terminal repeat (LTR), which is present in all retroviral genomes, HFV possesses a second promoter, the internal promoter (IP), located at the end of the env gene. HFV is nonpathogenic retrovirus with a wide tissue tropism commonly found in mammals and can infect cells from many vertebrate species and diverse tissue types. HFV vectors are able to transduce nondividing cells more efficiently than oncoretroviral vectors, have a large packaging capacity, and are not inactivated by human serum. These properties suggest that vectors derived from HFV may serve as safe and effective delivery vehicles in gene therapy.Based on an infectious molecular clone of HFV(pHSRVl), helper vector p Δ GP was successfully constructed by deletion of the gag and pol genes, substitution SV40 polyA signal for the 3' LTR of human foamy virus. Replication-defective vector pGPSNI-EGFP and helper vector p A GP were cotransfected into HIC cell line.Moreover, replication-defective vector pGPSNI-EGFP and helper vector p A GP were transfected into HIC cell line as a control, respectively. Using fluorescence microscopy, the cotransfected HIC cell with pGPSNI-EGFP and p A GP vectors strongly expressed green fluorescence protein (GFP) and the transfected HIC cell with replication-defective vector pGPSNI-EGFP weakly expressed green fluorescence protein, while the transfected HIC cell with helper vector p A GP did not completely express green fluorescence protein. The result showed not only that replication-defective vector pGPSNI-EGFP and helper vector pAGP from human foamy virus were successfully constructed, but also the 3' end sequence of human foamy virus' env gene (internal promoter, IP) had weak promoter activity and Bel protein promoted by IP transactivated both IP and the 5' LTR promoter of human foamy virus strongly.HFV vectors with their nonpathogenic nature and a wide tissue tropism may provide a safe and efficient means of transduced nondividing cells such as neurons. To investigate the potential of HFV-derived vector in gene therapy for neurological diseases, efficient foreign gene expression in cultured rat hippocampal neurons (HNs) was first demonstrated by successful EGFP transduction through a HFV vector bearing an EGFP expression cassette.Glutamic acid decarboxylase (GAD) is the speed limit enzyme of composing inhibitory transmitter GABA and the key factor in making of GABA . We sought to determine whether HFV containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of the central nervous system neurons. For this purpose, two novel replication-defective vectors plasmids pGPSNI-hGAD67 and pGPSNI-hGAD65 were constructed and contain two transcription units: one encoding the neomycine phosphotransferase gene and other encoding a single human GAD isoforms (hGAD67 or hGAD65). GAD expression in HNs cultures, as detected by isoform-specific immunocytochemistry, was observed in all treated groups. Intracellular and extracellular GABA from HNs were quantified by HPLC. Transduction of HNs by rdvGAD67 (m.o.i.=l) resulted in significant increases in the levels of intracellular GABA compared to rdvGFP (approximately 6-fold) ormock (approximately 4-fold) transduced cultures (p<0.005) and with rdvGAD65 resulted in a fewer increases in the levels of intracellular GABA than rdvGFP and mock transduced cultures. Transduction by rdvGAD67 resulted in a considerable increase (p<0.005) in basal release of GABA (non-stimulated extracellular GABA) as compared to rdvGFP or mock transduced cultures, but no increase in transduction by rdvGAD65. Depolarization of vector-transduced HNs cultures with 50 mM K+ significantly increase GABA release over basal levels in rdvGAD67 and also increase in rdvGAD65, rdvGFP, or mock transduced HNs cultures. These findings indicated for the first time that cultured rat HNs could be efficiently transduced by HFV vectors and GAD-expressing HFV vector has potential therapeutic value for the treatment of neurological diseases.In addition, efficient foreign gene expression in cultured rat astrocytes was first demonstrated by successful EGFP transduction through a HFV vector bearing an EGFP expression cassette. GAD expression in astrocytes cultures, as detected by isoform-specific immunocytochemistry, was observed in all treated groups. Significant amount of GABA, as determined by HPLC, were synthesized in cultures transduced with either GAD vector. GABA was undetectable in all cases for rdvGFP or mock-transduced cultures. GABA was tonically released from astrocytes transduced with rdvGAD67 or rdvGAD65, but a few increase could be detected after treatment of the cells with K+. No released GABA was detectable in all cases for rdvGFP or mock-transduced cultures. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurological disorders.