ABSTRACT
Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.
Subject(s)
Hypoxanthine Phosphoribosyltransferase/genetics , Simplexvirus/genetics , Animals , Brain/enzymology , Brain Diseases/enzymology , Brain Diseases/genetics , DNA/genetics , Genetic Vectors , Herpes Simplex/enzymology , Herpes Simplex/genetics , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Liver/enzymology , Mice , Mice, Inbred DBA , Nucleotide Mapping , RNA Probes , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Tumor Cells, CulturedSubject(s)
Hypoxanthine Phosphoribosyltransferase/genetics , RNA, Messenger/genetics , Animals , Brain/metabolism , Gene Expression Regulation, Enzymologic , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Mice , Mice, Inbred DBA , Mice, Transgenic , RNA, Messenger/metabolism , Simplexvirus/genetics , TransfectionABSTRACT
The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.
Subject(s)
Genetic Vectors , Hypoxanthine Phosphoribosyltransferase/genetics , Neurons/physiology , Simplexvirus/genetics , Transfection , Cloning, Molecular , DNA, Recombinant , HumansABSTRACT
Hypoxanthine-guanine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) functions in the purine-metabolic salvage pathway. Two clinical syndromes are associated with a deficiency in HPRT enzyme activity. Virtually complete deficiency leads to the Lesch-Nyhan syndrome, whereas partial deficiency results in hyperuricemia and severe gouty arthritis. Marked heterogeneity in the mutations leading to HPRT deficiency has been found. Mutant enzymes vary with respect to levels of HPRT immunoreactive protein, electrophoretic migration, kinetic properties and amino acid sequence. Analysis of DNA and RNA from patients with HPRT deficiency has revealed point mutations, an internal gene duplication and partial as well as complete gene deletions accounting for the various HPRT mutant enzymes.
