Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1994-08-26
1998-05-05
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4353201, 435325, C12N 1500, C12N 1563, C12N 506
Patent
active
057473072
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/GB93/00417, filed on Mar. 1, 1993.
FIELD OF THE INVENTION
This invention relates to vectors and their use in gene transfer. The vectors are based on retroviruses, adapted so that they cannot package their own RNA, and which can be used as infectious agents to transfer foreign genes, e.g. for somatic gene therapy.
BACKGROUND OF THE INVENTION
Retroviruses are classified in several ways. They are divided into various groups on the basis of their morphology. These groups are A,B,C and D type viruses. They are also classified as belonging to one of three subfamilies, namely oncoviruses, spumaviruses and lentiviruses.
Mason-Pfizer Monkey Virus (MPMV) is a D-type retrovirus first discovered in breast carcinoma tissue from a rhesus monkey. Despite this, and its classification into the oncovirus subfamily, it does not contain an identified oncogene and there is no evidence that it has oncogenic potential.
D-type viruses are distinguished from other retrovirus families such as the C-type viruses. The latter are characterised by capsid assembly at the cell membrane, and include viruses of the lentivirus group, e.g. Human Immunodeficiency Virus (HIV). Morphologically, in their core structure, D-type viruses also differ from B-type viruses such as Mouse Mammary Tumor Virus (MMTV). Thus the D-type viruses are completely distinct from these other types and as an example of this distinction they have specific signals in the form of amino-acid signal sequences in their assembled ICAP which facilitate transport of the ICAP to the cell membrane. For this reason and others they must be considered as a separate group with unique cis and trans-acting regulatory signals. MPMV is also distinguished from C-type viruses by the ability of the virus envelope to activate the human complement system. This leads to lysis of the virus.
Retroviruses are RNA viruses which replicate through a DNA proviral intermediate which is integrated in the genome of the infected host cell. The virion particle contains a dimer of positive-strand genomic RNA molecules. This genomic RNA is the full-length species transcribed from the proviral DNA by the host RNA polymerase II. A proportion of these full length RNAs which encode the gag and pol genes of the virus are translated by the host cell ribosomes, to produce the structural and enzymic proteins required for production of virion particles. The provirus also gives rise to a variety of smaller singly and multiply-spliced mRNAs coding for the envelope proteins and, in the case of more complex retroviruses, a group of regulatory proteins. The genomic (and subgenomic) RNA molecules are structurally similar to cellular mRNAs in having a 5' m.sup.7 G cap and a polyadenylated 3' tail.
A series of problems must be addressed for successful packaging of genomic RNA:
The full-length RNA must be packaged preferentially over the spliced viral messages as it is the only one carrying the full complement of genetic information for the next generation of virions. The virus must also specifically select the genomic RNA against the enormous quantity and variety of physically similar host cell mRNAs as, unlike many other viruses, retroviruses do not generally arrest host RNA synthesis. There must be a mechanism whereby genomic RNA to be packaged is recognised such that a proportion is either protected from being translated and transported to an assembly site or is associated with the gag precursor polyprotein for which it has just coded immediately after translation. Lastly, there is the stoichiometric problem of having to package the correct number of genomes in association with 3-4000 gag precursor proteins, adequate numbers of reverse transcriptase molecules, a protease, tRNA primers and, in some cases, multiple copies of regulatory proteins.
Packaging the genome thus entails problems of specificity of selection of RNA and also considerations of RNA compartmentalisation.
The virus overcomes these problems by the presence of cis-acting elements, i.e. "packaging signals", in the vi
REFERENCES:
Francki, R.I.B. et al. (1992) "Classification and Nomenclature of Viruses" Fifth Report of the Internatinal Committee on Taxonomy of Viruses, Archives of Virology Supplementum 2, pp. 293, 295, 297, 298.
Vile, Richard G. et al. (1992) "Identification of a Generalised Packaging Sequence for D-Type Retroviruses and Generation of a D-Type Retroviral Vector" Virology 189:786-791.
Hunter Eric
Lever Andrew Michael Lindsay
Robinson Douglas W.
Syngenix Limited
Wai Thanda
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