Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1999-10-15
2000-12-12
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435357, 435365, 4353201, 435455, 536 231, 536 241, C12P 2102
Patent
active
061597104
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates generally to methods of stabilizing unstable gene transcripts for use in, e.g., gene therapy, and specifically to the use of the HSV-1 LAT sequences in such methods.
BACKGROUND OF THE INVENTION
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus capable of forming latent infections for the lifetime of an individual. Upon stress the viral genome undergoes extensive transcription and replication leading to the production of viral proteins and infectious particles. Contrary to the acute initial infection or subsequent reactivation events, during latency transcription is restricted to a single diploid gene within the long repeat elements of the viral genome. Transcription of this gene generates a family of transcripts known as the latency-associated transcripts (LATs) (see FIG. 1) [Rock, D. L. et al., (1987), J. Virol., 61:3820-3826; Spivack, J. G., and N. W. Fraser, (1987), J. Virol., 61:3841-3847 (Spivack 1987); Stevens, J. G. et al, (1987), Science, 235:1056-1059; and Wechsler, S. L. et al, (1988), J. Virol., 62:4051-4058].
An 8.5 kb LAT (referred to as the minor LAT or MLAT based on its abundance) is postulated on the basis of in situ hybridization of infected tissues and the presence of a LAT promoter element mapping to its 5' end and of a polyadenylation signal near its 3' end. It is found in very low amounts in trigeminal ganglia of infected animals [Mitchell, W. J. et al, (1990), J. Gen. Virol., 71:125-132].
The most abundant LAT species is a 2 kb long transcript (referred to as 2.0 kb LAT), which does not appear to be polyadenylated [Devi-Rao, G. B. et al, (1991), J. Virol., 65:2179-2190; Nicosia, M. et al, (1994), 204:717-728 (Nicosia 1994); Spivack 1987; and Wagner 1988] and also lacks a cap at its 5' end which maps to a splice donor sequence GT [Krause, P. R. et al, (1990) J. Clin. Invest., 86(1):225-241; Krause, P. R. et al, (1991) J. Virol., 65:5619-5623; Spivack, J. G. et al, (1991), J. Virol., 65:6800-6810 (Spivack 1991); and Wagner 1988].
The 2.0 kb LAT is considered to be a unique class of genes, known as the .lambda. class [Spivack 1988]. It has been proposed that the 2.0 kb LAT is a stable intron derived from the larger 8.5 kb MnLAT RNA. Consistent with this, Farrell, M. J. et al, 1991, Proc. Natl. Acad. Sci. USA., 88:790-794 have shown that the 2.0 kb LAT RNA could be spliced out of a .beta.-galactosidase transcript containing the LAT sequences in transient transfections. additionally Wu, T. T. et al, 1996, J. Virol., 70:5962-5967 have recently shown that the majority of the 2.0 kb LAT transcript is in a non-linear structure most likely a lariat. However, the spliced exons of the putative primary transcript MLAT have never been detected.
Removal of a short intron in the 2.0 kb LAT leads to the production of a small variant of 1.5 kb in size. Both transcripts are routinely detectable by Northern hybridization [Rock et al, Spivack 1987, Stevens et al and Wechsler et al, cited above]. These RNAs are partially colinear and are thought to evolve by differential splicing. The 1.5 kb LAT is only observed during latency in neurons, whereas the 2.0 kb LAT is detectable in productive infections in tissue culture and animals with the kinetics of a late gene, as well as during latency [Spivack 1987; Spivack, J. G., and N. W. Fraser, (1988), J. Virol., 62:3281-3287 (Spivack 1988); Wagner, E. K. et al, (1988), J. Virol., 62:1194-1202 (Wagner 1988)].
Two promoters involved in the generation of the 2.0 kb LAT RNA have been identified. They are known as the Latency Active Promoter 1 (LAP 1) [Batchelor, A. H. and P. O'Hare, 1990, J. Virol., 64:3269-3279; Dobson, A. T. et al, 1989, J. Virol., 63:3844-3851; Zwaagstra, J. et al, 1991, Virol., 182:287-297; Zwaagstra et al, 1989, J. Gen. Virol., 70:2163-2169; Zwaagstra et al, 1990, J. Virol., 64:5019-5028] and the Latency Active Promoter 2 (LAP2) [Goins, W. F. et al, 1994. J. Virol., 68:2239-2252]. The LAP1 promoter, mapping to the 5' end of mLAT, is the promoter of this putative transcript. Specula
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Fraser Nigel W.
Krummenacher Claude F.
Zabolotny Janice M.
Ketter James
The Wistar Institute of Anatomy and Biology
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