Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-03-04
2002-04-30
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S325000, C435S455000, C435S069100
Reexamination Certificate
active
06379967
ABSTRACT:
The invention relates to a method of virus manipulation; means therefor and products thereof which have particular, but not exclusive, application in gene therapy.
Gene therapy of many diseases is now theoretically possible, as a result of recent advances in human genetics. The primary goal is the conversion of cell phenotype from a diseased to a normal state, through the delivery of trans-dominant acting genetic material. The conversion of this technology from cell culture systems to in vivo experimental models (and subsequently to the clinic) requires the development of new methods for efficient gene delivery in a controllable manner. It is becoming evident that whilst human genetics is moving at a rapid rate in the identification of disease-specific mutations, there is a relative lack of gene delivery system development. At present, there is a choice of either liposome, DNA aggregate or virus-based systems.
Liposome delivery is still very inefficient in DNA transfer (1), DNA aggregates formed between virus particles and charged materials such as polylysine do enhance DNA uptake (2) but standardisation of preparations is very difficult. Retrovirus and adenovirus vectors both have constraints in the size of heterologous DNA incorporated in the vector (3,4) and are unreliable in achieving long-term heterologous gene expression. Retroviruses integrate into the host genome but are difficult to produce as high titre stocks and have an inherently high rate of mutation through errors introduced during reverse transcription. Despite their broad cell tropism, adenoviruses induce a cell-mediated immune response and the nucleic acid is not stable long-term in infected cells (5).
Herpesviruses represent promising candidates for development as vectors, in part due to their ability to maintain their genome in cells in an episomal form which is blocked from replication. Their capacity for packaging heterologous DNA sequences is potentially >50 Kbp (6) and most are easy to manipulate in vitro. Herpes simplex derived vectors are likely to have some of the same problems as adenoviruses, in that the majority of the population already have a well-developed immune response to the virus. Other non-human herpesviruses which are capable of infecting human cells, however, should not suffer this disadvantage.
Herpesvirus saimiri (HVS) is a lymphotropic rhadinovirus (
&ggr;
2 herpesvirus) of squirrel monkeys (
Saimiri sciureus
). The virus may be routinely isolated from peripheral lymphocytes of healthy monkeys and causes no apparent disease in the species. The virus genome may be detected in an episomal form in T cells and genome transcription appears limited to three genes in the non-lytic (“latent”) state. The complete virus genome has been sequenced and shares many features in common with the human Epstein-Barr virus (EBV). The genetic organisation consists of a single unique coding region of DNA, 112.930 bp in length, flanked by a variable number of non-coding repeat sequences. There are 76 open reading frames, 60 of which have similarities with genes found in other herpesviruses (7). The remaining genes share sequence homology (at the level of protein) with human genes of known function, including complement control proteins, cell surface antigen CD59, cyclin D and G protein-coupled receptors (8, 9).
The virus has been divided into three distinct strains termed A, B and C based on their inability (A and B) or ability (C) to be oncogenic in certain other monkey species. C strains have the ability to transform human T cells to limited independent growth in vitro (10). This ability to transform cells is due to a gene termed STP (11) which has marked variability in protein sequence between strains such that only STP from C strains is able to transform cells (12). STP is not important for the normal lytic cycle of the virus or episomal maintenance and natural deletion mutants for this region of the virus genome exist (13); these strains are not oncogenic. Virus strains which lack this gene have been constructed which express selectable drug resistant markers (14). These viruses have been used to demonstrate that they are capable of infecting a wide range of human cell types, transferring heterologous genes with high efficiency and maintaining long-term expression in the absence of selective pressure. There is no evidence that this virus is able to produce any disease in man, although it is capable of infecting human cells. Thus it is likely that this virus represents a good starting point for the development of a non-replicating, safe vector for human cells. There is however a lack of basic understanding of how HVS replicates, particularly regarding transcriptional control and DNA replication.
Of all herpesviruses sequenced so far, HVS has the most homology with EBV. However the coding region is significantly smaller. Distinct gene blocks appear to be closely related between these two viruses, and indeed the herpesviruses in general. HVS differs from other herpesviruses due to the presence of certain genes which have not been identified in any other herpesvirus to date. Every virus vector in human trials to date has been disabled either through the deletion of genes which are non-essential for growth in culture or the deletion of essential genes and their provision in trans from helper cell lines. Extrapolation from the well studied herpesviruses allows us to predict that deletion of certain HVS membrane proteins will prevent cell-cell spread. Furthermore, the inactivation of proteins which control essential transcriptional switches, such as E1A in adenoviruses (17) and IE 175 in herpes simplex (18) will inevitably make such viruses replication incompetent. Thus, a major aim of this application is focussed on the construction of mutant viruses which are unable to activate early and late gene expression. The target genes are the two transcriptional control proteins which are the products of ORF 50 and 57, and likely to be essential for growth in tissue culture.
Published data (14) indicates that Strain 11/S4-derived viral vectors are only capable of limited growth in certain cell lines. Thus the need to delete, block or manipulate transcriptional control protein genes should only be necessary in cell lines that support viral replication. However, it may be desirable, in order to produce a virus for the purpose of gene delivery which one can use confidently, to produce a virus which is either unable to produce or which produces non functional transcriptional control proteins.
It is also another major aim of this application to identify genes which are non-essential for growth and then delete at least a part of at least one of these genes in order to facilitate the insertion of heterologous genetic material into the viral genome.
There currently exists a plasmid designed for recombination with herpesvirus saimiri which plasmid is designed to insert heterologous genetic material into the viral genome at a predetermined location, the location being the junction between the single unique coding region of DNA and a non-coding repeat sequence of herpesvirus DNA. However the plasmid is relatively inflexible in terms of what can be cloned into the viral genome. For example, there are few suitable restrictions sites and therefore the plasmid is not suitable for use commercially. We have therefore aimed in this application to identify non-essential genes for growth with a view to deleting at least a part of at least one of said genes with a view to providing artificial cloning sites for the insertion of large amounts of any selected heterologous genetic material. It will be apparent that the said deletion of non-essential genes and the subsequent insertion of heterologous genetic material will most advantageously be undertaken when large amounts of heterologous genetic material are to be inserted into the viral genome.
We aim in another aspect of our application to provide herpesvirus saimiri which has been manipulated so as to delete at least a part of at least one transcriptional control gene and, ideally,
Markham Alexander Fred
Meredith David Mark
Guzo David
Leffers, Jr. Gerald G.
Myers Bigel & Sibley & Sajovec
The University of Leeds
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