Use of spliceosome mediated RNA trans-splicing to confer cell selective replication to adenoviruses

#The present invention provides methods and compositions for conferring selective adenovirus mediated cell death on cells expressing a specific target precursor messenger RNA (selective target pre-mRNAs). The compositions of the invention include conditionally replicative adenoviruses that have been genetically engineered to express one or more pre-trans-splicing molecules (PTMs) designed to interact with one or more selective target pre-mRNA and mediate a trans-splicing reaction resulting in the generation of novel chimeric RNA molecules (chimeric RNA) capable of encoding an adenovirus specific protein(s). Adenovirus specific proteins include those proteins complementing an essential activity necessary for replication of conditionally replicative adenoviruses. Upon successful trans-splicing between the target pre-mRNA and the PTM, the adenovirus peptide(s) are expressed thereby providing the required complementing activity necessary for replication of the conditionally replicative adenoviruses. Such viral replication leads to cell lysis, thereby targeting selective destruction of infected cells.

#The methods and compositions of the invention may be used to treat a variety of different diseases where the goal is selective destruction of a specific cell type. For example, the present invention provides methods and compositions for conferring selective cell death on cancer cells expressing a specific target precursor messenger RNA molecules (cancer cell selective target pre-mRNAs). Such compositions include conditionally replicative adenoviruses that have been genetically engineered to express one or more pre-trans-splicing molecules (PTMs) designed to interact with one or more cancer cell selective target pre-mRNA and mediate a trans-splicing reaction resulting in the generation of novel chimeric RNA molecules (chimeric RNA) capable of encoding an adenovirus specific protein(s). Alternatively, the present invention may be utilized to confer selective cell death on cells infected with a pathogenic microorganism. In such instances, conditionally replicative adenoviruses are engineered to encode PTMs designed to interact with one or more target pre-mRNA encoded by the pathogenic microorganism, or induced within the cells of a subject infected with a pathogenic microorganism and encode an adenovirus polypeptide(s). Upon successful trans-splicing between the target pre-mRNA and the PTM, the adenovirus peptide(s) are expressed thereby providing the required complementing activity necessary for replication of the conditionally replicative adenoviruses.

#DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process referred to as splicing. In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing. Trans-splicing was first discovered in trypanosomes (Sutton & Boothroyd, 1986, Cell 47:527; Murphy et al., 1986, Cell 47:517) and subsequently in nematodes (Krause & Hirsh, 1987, Cell 49:753); flatworms (Rajkovic et al., 1990, Proc. Nat'l. Acad. Sci. USA, 87:8879; Davis et al., 1995, J. Biol. Chem. 270:21813) and in plant mitochondria (Malek et al., 1997, Proc. Nat'l. Acad. Sci. USA, 94:553). In the parasite Trypanosoma brucei, all mRNAs acquire a splice leader (SL) RNA at their 5′ termini by trans-splicing. A 5′ leader sequence is also trans-spliced onto some genes in Caenorhabditis elegans. This mechanism is appropriate for adding a single common sequence to many different transcripts.

#FIG. 1. Schematic representation of different trans-splicing reactions. (a) trans-splicing reactions between the target 5′ splice site and a PTM 3′ splice site; (b) trans-splicing reactions between the target 3′ splice site and a PTM 5′ splice site; (c) replacement of internal exon by double trans-splicing reaction in which the PTM carries both 5′ and 3′ splice sites. BD, binding domain, BP, branchpoint sequence, PPT, polypyrimidine tract and ss, splice site.

#FIG. 2. Schematic representation of adenoviruses as anti-cancer agents.

#FIG. 3. Schematic diagrams of PTM-mediated control of adenoviral replication.

#The present invention provides methods and compositions for conferring adenovirus mediated cell death on cells expressing a specific target precursor messenger RNA molecules. The target precursor messenger RNA molecules may be selectively expressed in cancer cells, or alternatively, the RNA molecules may be those encoded by infectious agents such as bacteria, parasites, fungi or viruses. Target pre-mRNAs also include those cellular pre-mRNAs induced during bacterial, parasitic, fungal or viral infection, or, pre-mRNAs wherein expression of said pre-mRNA is associated with a specific disease or disorder. The compositions of the invention include, for example, recombinant adenoviruses that have been genetically engineered to express pre-trans-splicing molecules (PTMs) designed to interact with one or more cancer cell selective target pre-mRNAs, or target pre-mRNAs encoded by an infectious agent and mediate trans-splicing reactions resulting in the generation of a novel chimeric RNA molecules (chimeric RNA) encoding adenovirus specific protein(s) capable of rescuing the conditionally replicative adenovirus. Specifically, the PTMs of the invention are designed to encode adenovirus specific proteins that are required for replication of conditionally replicative adenovirus thereby providing for complementation of said adenovirus. The methods and compositions of the invention may be used to target a lytic adenovirus infection to cancer cells or cells infected with a pathogenic agent thereby providing a method for selective destruction of cancer cells or cells infected with an infectious agent. In addition, the recombinant adenoviruses may be designed to express PTM molecules that encode for diagnostic or therapeutic polypeptides. The polypeptides may be expressed independently, or alternatively, as adenovirus fusion proteins. Such recombinant conditionally replicative adenoviruses permit targeting of a therapeutic or diagnostic polypeptide to a cell expressing a selective pre-mRNA.

#The compositions of the invention include recombinant conditionally replicative adenoviruses that have been genetically engineered to express one or more PTMs designed to interact with one or more selective target pre-mRNA molecule such as, for example, cancer cell selective target pre-mRNA, target pre-mRNA molecules encoded by an infectious agent, target cellular pre-mRNAs induced by an infectious microorganism, or target pre-mRNAs where the expression of said pre-mRNA is associated with a disease or disorder. Such RNAs are designed to mediate trans-splicing reactions resulting in the generation of novel chimeric RNA molecules (chimeric RNAs). The novel chimeric RNA is designed to encode a complementing adenovirus protein(s) capable of rescuing the defect of the conditionally replicative adenovirus. Such rescue leads to a lytic adenovirus infection resulting in cell lysis. The compositions of the invention provide a means for conferring selective adenovirus mediated cell death on cells expressing a specific target pre-mRNA. The conditionally replicative adenoviruses of the invention are designed to encode PTMs comprising (i) one or more target binding domains that targets binding of the PTM to a specific pre-mRNA target (ii) a 3′ splice region that includes a 3′ splice acceptor site and/or 5′ splice donor site; and (iii) a nucleotide sequence capable of encoding at least one adenovirus protein necessary for replication of adenovirus. In addition to adenovirus proteins, the PTMs may include sequences capable of encoding diagnostic or therapeutic polypeptides. Such polypeptides may be expressed as adenovirus fusion proteins, wherein the adenovirus portion of the fusion protein retains its ability to provide complementing activity. Alternatively, PTMs may be engineered to independently express the diagnostic or therapeutic polypeptide.

#In some instances, the PTMs of the invention may further comprise one or more spacer regions that separate the RNA splice site from the target binding domains and/or a safety sequence. The structure of PTMs is described in detail in U.S. Pat. Nos. 6,013,487, 6,083,702, 6,280,978, and in co-pending U.S. patent application Ser. Nos. 09/756,095, 09/756,096, 09/756,097 the disclosures of which are incorporated by reference herein.