Retrovirus vectors have received considerable attention as gene transfer vehicles for potential use in human therapy of single gene disorders, including Lesch-Nyhan syndrome. Lesch-Nyhan syndrome is caused by the deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT), and is characterized by behavioral alterations, including self-injurious behavior and mental retardation. The pattern of neurological abnormalities suggests potential involvement of the basal ganglia, where high levels of HPRT enzyme and mRNA have been detected in normal brain. Recombinant murine retroviruses have been developed as gene transfer vehicles to deliver functional gene products, such as HPRT, into cells that contain nonfunctional or deficient product. Recently, my laboratory isolated an infectious molecular clone of a type D serogroup 2 simian retrovirus (SRV; D2/RHE/OR) found within Asian macaques. This retrovirus exhibits broad tropism within the nonhuman primate, and has been found to nonproductively infect the brain and nervous system. Sequence analysis of this molecular clone, and of several other virus variants which exhibit distinct differences in targeted cells, has allowed us to define the genetic structure of the envelope (env) glycoprotein gene and to develop a structural model of simian retrovirus-cell interaction. The overall goal of this project is to develop the type D retrovirus as a gene transfer/therapy vehicle. As the ultImate goal of this research, but in part beyond the immediate scope of our two-year proposal, we will assess the ability of the type D SRV gene transfer vehicle to infect and express reporter markers in the brain, and then utilize this vector as a potential gene therapy vehicle for the expression of HPRT in the Lesch-Nyhan syndrome nonhuman primate model.
The specific aims of this application are: 1) Identification of the psi packaging signal for the serogroup 2 simian retroviruses: We have currently sequenced the D2/RHE/OR envelope glycoprotein gene in its entirety, and portions of the 3' long terminal repeat (LTR) region and reverse transcriptase (pol) gene. In order to develop simian retrovirus packaging cell lines and to identify this virus' primary psi packaging signal and other facultative packaging sites, we will need to subclone the complete D2/RHE/OR genome into a low-copy number plasmid vector and sequence its gag gene and 5' LTR. This sequence information is required to create packaging cell vectors lacking the psi packaging signal. The psi packaging signal has been localized to a 624 bp region in the gag gene contained within a related simian retrovirus, the Mason-Pfizer monkey virus, and should be readily identified in our D2/RHE/OR molecular clone by sequence similarity analyses. 2) Construction of simian retrovirus packaging cell line and gene transfer vehicle: To obtain packaging cell vectors, we will construct two partial D2/RHE/OR plasmid recombinants containing distinct retroviral gene regions (gag, pro, and pol on one recombinant, and env on the other recombinant; Experiment A). In addition to the retroviral structural genes, these recombinants will contain a modified D2/RHE/OR 5' LTR, an SV40 polyadenylation signal in place of a deleted 3' LTR, and a neo positive selection marker placed outside the retroviral transcription unit. The gag-pro-pol recombinant will have the psi packaging signal deleted. Both recombinants will be cotransfected into CV-1 cells (Monkey kidney cells) to create a simian retrovirus packaging cell line. Cotransfection of molecular clones containing these modifications should dramatically reduce the possibility of recombination and regeneration of wild-type, replication-competent viruses. In addition, to obtain the retrovirus gene transfer vehicle, we will delete the structural genes of the simian retrovirus molecular clone and substitute a cassette including the psi packaging signal, a reporter gene (eg: beta-galactosidase), and a multiple cloning site for potential introduction of other secondary therapeutic genes (e.g.: HPRT gene; Experiment B). This retrovirus vehicle will then be transfected into the packaging cell line to generate replication-defective virus transduction particles. To begin testing the recombinant virus' ability to infect primate targets, with the ultimate aim of generating gene transfer vehicles for both nonhuman and human primates, we will conduct in vitro infections of recombinant viruses with defined monkey cells (e.g.: Vero cells) and with established human cell lines (e.g.: human neuroblastoma cell line SKN-MC and others). 3) Nervous system infection of nonhuman primates with recombinant retroviruses and assessment of reporter gene expression within the basal ganglia: Using high-titer sources of recombinant simian retrovirus, we will infect the basal ganglia of nonhuman primates by stereotactic injection, and following autopsy, assess the expression of the beta- galactosidase reporter gene in the surrounding tissue. Beyond the scope of this application, but within the overall future goals of this research program, will be the use of a described Lesch-Nyhan nonhuman primate model, and its potential therapy by a HPRT-containing simian retrovirus vector.
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