Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1994-07-26
2003-09-02
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S069100, C435S325000, C435S091400, C435S455000
Reexamination Certificate
active
06613749
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the packaging of material for delivery, for example in gene therapy and other situations.
2. Description of Related Art
Gene transfer, as a means to effect gene therapy, is becoming increasingly important. Numerous studies have been carried out that seek to harness the inherent ability of viruses to infect eukaryotic cells in order to introduce a selected gene into a suitable recipient host (as reviewed: Anderson, 1992; Morgan and Anderson, 1993; Mulligan, 1993). Adenoviruses have proved particularly attractive for this purpose in that their genome can be manipulated to incorporate up to about 8 kbp of foreign DNA and, unlike retroviral vectors, transduced genes, can be expressed in non-dividing cells. One particular handicap shared by all the present virus vectors, however, is that they introduce unwanted viral genetic information alongside the gene of interest into the recipient host. The disadvantages of this unwanted viral material remain to be determined fully, but there is a great need for means to carry out improved genetic transfer without introduction of unwanted viral genes into target cells.
Physical, non-viral gene transfer methods such as chemical and mechanical techniques and membrane fusion-mediated transfer via liposomes have been used (Morgan and Anderson, 1993). However, the efficiency of transfer, in terms of expression of the genetic material transferred, is low. In other words, these methods are inefficient at transferring genetic material into cells in a stable manner so that the material is biologically functional.
The empty capsids of papovaviruses such as the mouse polyoma virus have received attention as possible vectors for gene transfer.
Barr et al, 1979, first described the use of polyoma empty particles when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particle was protected from the action of pancreatic DNase. Slilaty and Aposhian, 1983, describe the use of those reconstituted particles for transferring a transforming polyoma DNA fragment to rat FIII cells. The empty capsids and reconstituted particles consist of all three of the polyoma capsid antigens VP1, VP2 and VP3 and there is no suggestion that pseudocapsids consisting of only the major capsid antigen VP1, could be used in genetic transfer.
Montross et al. 1991, described only the major capsid antigen, the cloning of the polyoma virus VP1 gene and its expression in insect cells. Self-assembly of empty pseudocapsids consisting of VP1 is disclosed, and pseudocapsids are said not to contain DNA. It is also reported that DNA inhibits the in vitro assembly of VP1 into empty pseudocapsids, which suggests that said pseudocapsids could not be used to package exogenous DNA for transfer to host cells. The results of Sandig et al. 1993, showed that empty capsids incorporating exogenous DNA could transfer DNA in a biologically functional manner to host cells only if the particles consisted of all three polyoma capsid antigens VP1, VP2 and VP3. Pseudocapsids consisting of VP1 were said to be unable to transfer the exogenous DNA so that it could be expressed in the host cells. We now know that this was probably because Ca
2+
ions were not included in the medium in which the pseudocapsids were prepared. Haynes et al (1993) J. Virol., 67, 2486-2495) discuss the effect of calcium ions on empty VP1 pseudocapsid assembly. Hagensee et al (1993) J. Virol., 67, 315-322 disclosed the production of L1 and L1/L2 human papillomavirus capsids, but did not suggest their use to carry exogenous material.
BRIEF SUMMARY OF THE INVENTION
The present invention aims to overcome the above problems associated with known viral and non-viral methods of exogenous material transfer by providing a method involving the use of a pseudocapsid consisting of a papovavirus major capsid antigen to transfer exogenous material to a host cell wherein that material is biologically functional.
The invention also aims to provide a pseudocapsid suitable for use in the above transfer methods, and a method of making a pseudocapsid.
The invention also aims to provide a pharmaceutical composition comprising the pseudocapsid of the invention and a pharmacologically acceptable carrier.
According to the invention there is provided a method of transferring material into a host cell comprising providing a pseudocapsid formed from papovavirus major capsid antigen and excluding minor capsid antigens, which pseudocapsid has exogenous material associated therewith; and treating the host cell with the pseudocapsid so that the material is taken up by the cell and is biologically functional in that cell.
By “pseudocapsid” we mean a structure in which the only papovavirus capsid antigen is the major capsid antigen without minor capsid antigens, which structure is hollow so that it can contain exogenous material.
The term “papovavirus” defines a general family of viruses including polyoma virus (a mouse, virus), simian virus 40 (SV40), human variants (such as BK and JC) and papillomaviruses including human and bovine variants and other members. In each case, there is a major capsid antigen and one or more minor capsid antigens. For example, in papillomavirus the major antigen is L1 and the minor antigen is L2. In the present invention, the “pseudocapsids” are formed from the major capsid antigen and not the minor antigen(s).
The term “major capsid antigen” includes conservative variants thereof capable of assembling into a pseudocapsid and preferably having an antigenic determinant in common with the wild type antigen. The capsid antigen can be a hybrid of polyomavirus major capsid antigens, for example a hybrid of human BK virus major capsid antigen and mouse polyoma virus VP1.
By the exogenous material being “associated with” the pseudocapsid, we mean that the material is protected thereby. For example, exogenous DNA will be protected from degradation by DNases such as DNaseI, and exogenous protein will be protected from proteases. The exogenous material can be enclosed within an empty pseudocapsid or otherwise wrapped up with the capsid antigen.
The inventors have demonstrated that a simplified carrier consisting of a pseudocapsid involving only the major papovaviral capsid antigen can package and protect exogenous DNA, and transfer it into cells in a manner that allows for functional expression. A well-characterised gene, an efficiently transforming mutant form of the mouse polyoma virus middle T-antigen (MT), designated dl8MT (Griffin and Maddock, 1979), was chosen to show the worth of this system. The dl8-derived antigen possesses distinct physical and biological properties, which allow it to be easily assayed and distinguished from any wild type viral gene product that might have inadvertently contaminated recipient cells.
Papovaviruses have a broad host range and permissive cells include mammalian cells, such as rodent and human cells (Ponten, 1971). The pseudocapsids of the invention share this broad host range and can therefore be utilised in the transfer of exogenous material to a wide variety of cells.
The inventors have demonstrated that the pseudocapsids can successfully transfer exogenous genetic material in human cells.
Preferred host cells include mammalian cells, in particular rodents such as mice and rats, and human cells. Especially preferred human cells for in vivo gene transfer in accordance with the invention are lung cells.
Physical, non-viral gene transfer methods such as chemical and mechanical techniques and membrane fusion-mediated transfer via liposomes have been recently reviewed and assessed (Morgan and Anderson, 1993). The pseudocapsid (in the preferred embodiment involving genetic material) approach of the present invention resembles these procedures in that only the DNA intended for study, and no unwanted viral information, is introduced into cells. Moreover, the efficiency of functional gene transfer achieved by the method of the invention has been found to be between fifty and a hundr
Forstová Jitka
Griffin Beverly Elayne
Krauzewicz Nina Sheila
Holland & Knight LLP
Imperial College Innovations Limited
Nguyen Dave T.
LandOfFree
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