Title:  Methods and means for inducing apoptosis by interference in RNA processing

United States Patent:  6,620,925

Issued:  September 16, 2003

Inventors:  Noteborn; M. H. M. (Leiderdorp, NL)

Assignee:  Leadd B.V. (Leiden, NL)

Appl. No.:  452846

Filed:  December 3, 1999

Abstract

The invention relates to activation of apoptosis by means of interference with the function of snRNPs and hnRNP-like compounds. Also the invention relates to anti-tumor therapies with compounds, which negatively interfere with snRNPs and hnRNP-like compounds leading to induction of apoptosis, resulting in the elimination of tumor cells. Also the invention relates to therapies for diseases related to aberrant apoptosis induction, such as auto-immune diseases.

Description of the Invention

The present invention relates to the field of apoptosis, as well as to the field of cancer diagnosis and treatment and diagnosis and treatment of auto-immune diseases and other diseases. In particular the invention relates to improved methods and means for inducing apoptosis in cells to be eliminated. In particular the invention relates to novel means and methods for inducing apoptosis by interfering with the RNA processing machinery of a cell. In particular it relates to inhibiting or modifying the function of RNA-protein complexes involved in RNA processing such as snRNPs and hnRNP's. Both complexes are shown herein to be components of the apoptotic pathway that can be induced by chicken anemia virus proteins VP2 and/or VP3 (also called apoptin), both the hnRNP-like and snRNPs compounds are shown to associate to CAV-derived proteins Apoptin and VP2, which both are known to be involved in the apoptotic process. Apoptin and VP2 as stated, are proteins originally found in chicken anemia virus (CAV; Noteborn et al., 1991; apoptin was originally called VP3. The apoptotic activity of these proteins was discovered by the group of the present inventors (Noteborn et al., 1994, 1997).

Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or malignant cells (Earnshaw, 1995, Duke et al., 1996). Apoptosis characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into domain-sized fragments, in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighbouring cells and/or macrophages will rapidly phagocytose these dying cells (Wyllie et al., 1980, White, 1996). Cells grown under tissue-culture conditions and cells from tissue material can be analysed for being apoptotic with agents staining DNA, as e.g. DAPI, which stains normal DNA strongly and regularly, whereas apoptotic DNA is stained weakly and/or irregularly (Noteborn et al., 1994, Telford et al., 1992).

The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995, White 1996, Levine, 1997). Changes in the cell survival rate play an important role in human pathogenesis, e.g. in cancer development, which is caused by enhanced proliferation but also by decreased cell death (Kerr et al., 1994, Paulovich, 1997) A variety of chemotherapeutic compounds and radiation have been demonstrated to induce apoptosis in tumor cells, in many instances via wild-type p53 protein (Thompson, 995, Bellamy et al., 1995, Steller, 1995, McDonell et al., 1995).

Many tumors, however, acquire a mutation in p53 during their development, often correlating with poor response to cancer therapy. Transforming genes of tumorigenic DNA viruses inactivate p53 by directly binding to it (Teodoro, 1997). An example of such an agent is the large T antigen of the tumor DNA virus SV40. For several (leukemic) tumors, a high expression level of the proto-oncogene Bcl-2 or Bcr-abl is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry 994, Sachs and Lotem, 1997).

For such cancers (representing more than half of the tumors) alternative anti-tumor therapies are under development based on induction of apoptosis independent of p53 (Thompson 1995, Paulovich et al., 1997). One has to search for the factors involved in induction of apoptosis, which do not need p53 and/or can not be blocked by Bcl-2/Bcr-abl-like anti-apoptotic activities. These factors might be part of a distinct apoptosis pathway or being (far) downstream to the apoptosis inhibiting compounds.

Apoptin is a small protein derived from chicken anemia virus (CAV; Noteborn and De Boer, 1995, Noteborn et al., 1991, Noteborn et al, 1994), which can induce apoptosis in human malignant and transformed cell line, but not in untransformed human cell lines. In vitro, apoptin fails to induce programmed cell death in normal lymphoid dermal, epidermal, endothelial and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. (Danen-van Ooschot, 1997 and Noteborn, 1996). Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells.

In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells i.e. characterized by hyperplasia, metaplasia or dysplasia, it was located in the nucleus, suggesting that the localization of apoptin is related to its activity (Danen-van Oorschot et al. 1997).

Apoptin-induced apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995a), and cannot be blocked by Bcl-2, Bcr-abl (Zhuang et al., 199S), the Bcl-2-associating protein BAG-1 and not by the caspase-inhibitor cowpox protein CrmA (Danen-Van Oorschot, 1997a, Noteborn, 1996).

Therefore, apoptin is a potent agent for the destruction of tumor cells, or other hyperplasia, metaplasia or dysplasia which have become resistant to (chemo)therapeutic induction of apoptosis, due to the lack of functional p53 and (over)-expression of Bcl-2 and other apoptosis-inhibiting agents (Noteborn et al., 1997).

The fact that apoptin does not induce apoptosis in normal transformed human cells, at least not in vitro, suggests that a toxic effect of apoptin treatment in vivo will be very low. Noteborn et al. (1997) have provided evidence that adenovirus expressed apoptin does not have an acute toxic effect in vivo. In addition, in nude mice it was shown that apoptin has a strong anti-tumor activity.

It appears, that even pre-malignant, minimally transformed cells, may be sensitive to the death-inducing effect of apoptin. In addition, Noteborn and Zhang (1997) have shown that apoptin-induced apoptosis can be used as diagnosis of cancer-prone cells and treatment of cancer-prone cells.

Knowing that apoptin is quite safe in normal cells, but that as soon as a cell becomes transformed and/or immortalized (the terms may be used interchangeable herein) the present inventors designed novel menans and methoids for induction of apoptosis based on the identification of compounds involved in the apoptin-induced apoptotic cascade. These compounds are factors of an apoptosis pathway, which is specific for transformed cells, Therefore, these proteins are very important compounds in new treatments and diagnosis for diseases related with aberrancies in the apoptotic process, such as cancer, and (auto-)immune diseases.

A group of proteins found to be associated with apoptin is the family of hnRNP-like proteins.

The invention provides an apoptin-associating hnRNP-like protein, which is needed for RNA processing. When apoptin associates with such proteins it interferes with normal RNA processing, thus leading to apoptosis.

The invention thus further provides a method for inducing apoptosis through interference with hnRNP-like proteins (interchangeably referred to as hnRNP or hnRNP-like proteins) or other parts of hnRNP's.

The invention provides an anti-tumor therapy based on the interference with hnRNP-like proteins or other parts of hnRNP's.

As an additional mechanism hnRNP can shuttle apoptin or apoptin-like compounds to the nucleus where these compounds can induce apoptosis.

The invention thus provides hnRNP as mediator of apoptin-induced apoptosis, which is tumor-specific.

The present inventors have also shown a colocalization of VP2 with snRNP's another compound also involved in RNA processing.

The invention provides a VP2-associating snRNP-like protein or component which Is needed for RNA processing The invention further provides a method for inducing apoptosis through interference with snRNP-like Proteins or components (interchangeably referred to as snRNP or snRNP-like proteins).

The invention provides an anti-tumor therapy based on the interference with snRNP-like proteins. The invention provides snRNP as mediator of VP2-induced apoptosis.

The invention further provides a method for inducing apoptosis through interference with hnRNP-like and snRNP-like proteins.

The invention provides an anti-tumor therapy based on the interference with either or both snRNP- and hnRNP-like proteins.

The invention provides hnRNP and snRNP as mediators of VP2-induced apoptosis.

More in detail the invention provides a recombinant and/or isolated nucleic acid molecule encoding a member of the family of hnRNP proteins involved in RNA processing comprising at least a functional part of the sequence of FIG. 1 or a sequence having at least 60, preferably 70, preferably 80, more preferably 90% homology with said sequence. In cells where a particular hnRNP is not used for RNA processing such hnRNP activity can be used to shuttle apoptotic agents such as apoptin to the nucleus. It is then preferred to have such activity in an expression vector. hnRNP (-like) activity is defined as any molecule directly or indirectly providing the same kind of activity as an hnRNP or an hnRNP-like protein.

Such a vector preferably also encodes apoptotic activity, preferably apoptin-like activity which is defined analogous to hnRNP-like activity.

In this definition functional equivalents and/or fragments of apoptin are also encompassed.

In the case where hnRNP's are involved in RNA processing these compounds can be inhibited by apoptin-like activity, but also by for instance antisense molecules for hnRNP components. The invention thus also provides a recombinant and/or isolated nucleic acid molecule encoding an antisense recombinant molecule which can hybridize with a recombinant acid molecule according to claim 1. Preferably again such a molecule is present in an expression vector.

Apoptosis is preferably induced in a gene therapy setting, so that it is preferred to deliver all vectors to cells making use of gene delivery vehicle. Gene delivery vehicle are known in the art and our capable of transporting nucleic acid molecules of interest to cells. They include recombinant viruses (such as adenoviruses and retroviruses) as well as polymers and liposomes and the like.

It is preferred to also block the snRNP involvement in RNA processing. This can be done by VP2 (or VP2-like activity (same definition as hnRNP-like activity)) or by a further antisense molecule hybridizing with a nucleic acid molecule encoding a snRNP component.

Both options are provided by the present invention. The invention thus provides an expression vector encoding an antisense molecule for a nucleic acid encoding a component of an snRNP, preferably together with an hnRNP antisense molecule.

The invention also provides a method for identifying apoptotic agents comprising the use of nucleic acid molecules encoding members of the hnRNP-like family and the snRNP-like family.

Apoptotic agents identified by such a route are also considered part of this invention. These agents will typically be hnRNP antagonists or snRNP antagonists of which apoptin and VP2 are the first examples.

The most preferred method of inducing apoptosis is using antagonists to both snRNP and hnRNP, but often singel antagonists will suffice.

Claim 1 of 16 Claims

What is claimed is:

1. A recombinant and/or isolated nucleic acid molecule encoding a member of the family of hnRNP proteins involved in RNA processing, said recombinant and/or isolated nucleic acid molecule comprising:

the sequence of SEQ ID NO: 1.

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