Integrin-targeting vectors having transfection activity

Details Integrin-targeting vectors having transfection activity Integrin-targeting vectors having transfection activity

1999-11-29

2002-10-01

Crouch, Deborah (Department: 1632)

Coin handling

Stacker

C435S455000, C435S458000, C435S320100, C435S325000, C424S450000

Reexamination Certificate

active

06458026

ABSTRACT:

The present invention relates to an improved integrin-targeting vector that has enhanced transfection activity.
Gene therapy and gene vaccination are techniques that offer interesting possibilities for the treatment and/or prophylaxis of a variety of conditions, as does anti-sense therapy. Such techniques require the introduction of a DNA of interest into target cells. The ability to transfer sufficient DNA to specific target cells remains one of the main limitations to the development of gene therapy, anti-sense therapy and gene vaccination. Both viral and non-viral DNA delivery systems have been proposed. In some cases RNA is used instead of DNA. Receptor-mediated gene delivery is a non-viral method of gene transfer that exploits the physiological cellular process, receptor-mediated endocytosis to internalise DNA. Receptor-mediated non-viral vectors have several advantages over viral vectors. In particular, they lack pathogenicity; they allow targeted gene delivery to specific cell types and they are not restricted in the size of nucleic acid molecules that can be packaged. Gene expression is achieved only if the nucleic acid component of the complex is released intact from the endosome to the cytoplasm and then crosses the nuclear membrane to access the nuclear transcription machinery. However, transfection efficiency is generally poor relative to viral vectors owing to endosomal degradation of the nucleic acid component, failure of the nucleic acid to enter the nucleus and the exclusion of aggregates larger than about 150 nm from clathrin coated vesicles.
Integrins are a super-family of heterodimeric membrane proteins consisting of several different &agr; and &bgr; subunits. They are important for attachment of cells to the extracellular matrix; cell-cell interactions and signal transduction. Integrin-mediated cell entry is exploited for cell attachment and entry by a number of intracellular pathogens including
Typanosoma cruzi
(Fernandez et al., 1993), adenovirus (Wickham et al., 1993), echovirus (Bergelson et al., 1992) and foot-and-mouth disease virus (Logan et al., 1993) as well as the enteropathogen Y. pseudotuberculosis (Isberg, 1991). Egg-sperm fusion is also integrin mediated. Intensive study of the invasin-integrin mediated internalisation process of
Yersinia pseudotuberculosis
demonstrated that, for efficient cell entry, integrin-binding ligands should have a high binding affinity and a non-polar distribution (Isberg, 1991). Integrin-mediated internalisation proceeds by a phagocytic-like process allowing the internalisation of bacterial cells one to two micrometers in diameter (Isberg, 1991). Targeting of non-viral vectors to integrins, therefore, has the potential to transfect cells in a process that mimics infection of cells by pathogens and avoids the size limitation imposed by clathrin-coated vesicles in receptor-mediated endocytosis.
A further advantage of integrin-mediated vectors is that a large number of peptide ligands for integrin receptors have been described, including sequences derived from natural protein ligands [Verfaille, 1994 #635; Wang, 1995 #645; Staatz, 1991 #539; Pierschbacher, 1984 #314; Massia, 1992 #86, Clements et al. 1994 & Lu et al, 1993] or selected from phage display libraries (Koivunen et al. 1995; 1993; 1994; O'Neil et al. 1992; Healy et al 1995; Pasqualani et al. 1995).
The conserved amino acid sequence arginine-glycine-aspartic acid (RGD) is an evolutionarily conserved feature of many, but not all, natural integrin-binding ligands such as extracellular matrix proteins and viral capsids. Peptides, particularly those containing cyclic-RGD domains can also bind integrins. Peptides containing cyclic-RGD domains are particularly suitable ligands for vectors since they bind to integrins with higher affinities than linear peptides (Koivunen et al. 1995). Hart et al. have demonstrated previously that multiple copies of a cyclic RGD peptide displayed in the major coat protein subunit of fd filamentous phage particles, approximately 900 nm in length, are internalised efficiently by cells in tissue culture in an integrin-mediated manner (Hart et al., 1994). The phage particles were probably internalised by a phagocytic-like process as their size would exclude them from endocytosed vesicles (Hart et al., 1994).
The cyclic RGD-containing peptide GGCRGDMFGCGG[K]
16
[SEQ.ID.NO.:1] was synthesised with a sixteen-lysine tail for complex formation with plasmid DNA (Hart et al., 1995). Significant levels of integrin-mediated gene expression were achieved in epithelial cell lines with the vector GGCRGDMFGCG[K]
16
[SEQ.ID.NO.:2] (Hart et al., 1995) and the vectors GGCRGDMFGC[K]
16
[SEQ.ID.NO.:3] (WO96/15811). A similar peptide [K]
16
GACRGDMFGCA [SEQ.ID.NO. :4], which has the sixteen-lysine domain at the N-terminus and which is easier to synthesise than the prototype peptide (WO96/15811 and Hart et al., 1997) generated better transfection levels. Integrin mediated gene expression was generally achieved at levels of about 1 to 10%. The presence of chloroquine in the transfection medium gave some enhancement of transfection in some but not all cell lines tested.
The present invention is based on the surprising observation that inclusion of a lipid component in the oligolysine/-peptide/DNA complex increases levels of transfection of DNA from about 1 to 10% to about 50 to almost 100%. Not only is the level of transfection increased dramatically but, contrary to previous experience, the increase is observed in all cell lines tested, including endothelial, epithelial and tumour cell lines.
The present invention provides a complex that comprises
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component,
(iii) a polycationic nucleic acid-binding component, and
(iv) a lipid component.
The complex is a transfection vector.
The nucleic acid may be obtained from natural sources, or may be produced recombinantly or by chemical synthesis. It may be modified, for example, to comprise a molecule having a specific function, for example, a nuclear targeting molecule. The nucleic acid may be DNA or RNA. DNA may be single stranded or double stranded. The nucleic acid may be suitable for use in gene therapy, in gene vaccination or in anti-sense therapy. The nucleic acid may be or may relate to a gene that is the target for particular gene therapy or may be a molecule that can function as a gene vaccine or as an anti-sense therapeutic agent. The nucleic acid may be or correspond to a complete coding sequence or may be part of a coding sequence.
Alternatively, the nucleic acid may encode a protein that is commercially useful, for example industrially or scientifically useful, for example an enzyme; pharmaceutically useful, for example, a protein that can be used therapeutically or prophylactically as a medicament or vaccine; or diagnostically useful, for example, an antigen for use in an ELISA. Host cells capable of producing commercially useful proteins are sometimes called “cell factories”.
Appropriate transcriptional and translational control elements are generally provided. For gene therapy, the nucleic acid component is generally presented in the form of a nucleic acid insert in a plasmid or vector. In some cases, however, it is not necessary to incorporate the nucleic acid component in a vector in order to achieve expression. For example, gene vaccination and anti-sense therapy can be achieved using a naked nucleic acid.
The nucleic acid is generally DNA but RNA may be used in some cases, for example, in cancer vaccination. The nucleic acid component is referred to below as the plasmid component or component “ED”.
The integrin-binding component is any component that is capable of binding specifically to integrins found on the surface of cells. The integrin-binding component may be a naturally occurring integrin-binding ligand, for example, an extra-cellular matrix protein, a viral capsid pro

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