Proteome Sciences. AGM Statement.

In continuation of 23855 / 23865 / 23868 / 23903.

maxwellsdemon 1 Mar'05 - 15:52 - 7943 of 23903 (premium)


I just found this patent application although it was published some time ago. I am pretty certain it has not been posted on ADVFN before.

United States Patent Application 20040132181
Kind Code A1
Mitchell, Lloyd G. ; et al. July 8, 2004

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Trans-splicing mediated photodynamic therapy


Abstract
The present invention provides methods and compositions for conferring selective death on cells expressing a specific target precursor messenger RNA (selective target pre-mRNA). The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) expressed within a cell and mediate a trans-splicing reaction resulting in the generation of a novel chimeric mRNA molecule (chimeric mRNA) capable of encoding a light producing protein or enzyme. Cell death is further mediated by the presence of a photosensitizer which upon photoactivation produces cytotoxicity.


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Inventors: Mitchell, Lloyd G.; (Bethesda, MD) ; Otto, Edward; (Great Falls, VA) ; Merril, Carl R.; (Bethesda, MD)
Correspondence Name and Address: BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112


Serial No.: 658617
Series Code: 10
Filed: September 9, 2003

U.S. Current Class: 435/325
U.S. Class at Publication: 435/325
Intern'l Class: C12N 005/06; C12Q 001/68



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Claims

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We claim:

1. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; (b) a 3' splice region comprising a 3' splice acceptor site; (c) a spacer region that separates the 3' splice region from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

2. The cell of claim 1 wherein the 3' splice region further comprises a branch point and a pyrimidine tract.

3. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; (b) a 5' splice site; (c) a spacer region that separates the 5' splice site from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

4. The cell of claim 1 or 2 wherein the nucleic acid molecule further comprises a 5' donor site.

5. A method of producing a chimeric mRNA molecule in a cell wherein said chimeric molecule expresses a light producing protein or enzyme comprising contacting a target pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; (b) a 3' splice region comprising a 3' splice acceptor site; (c) a spacer region that separates the 3' splice region from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric mRNA within the cell.

6. The method of claim 5 wherein said 3' splice region further comprises a branch point and a pyrimidine tract.

7. A method of producing a chimeric mRNA molecule in a cell wherein said chimeric molecule expresses a light producing protein or enzyme comprising contacting a target pre-mRNA expressed within the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; (b) a 5' splice site; (c) a spacer region that separates the 5' splice site from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric mRNA within the cell.

8. The method of claim 5 or 6 wherein the nucleic acid molecule further comprises a 5' donor site.

9. A nucleic acid molecule comprising: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within a cell; (b) a 3' splice region comprising a 3' splice acceptor site; (c) a spacer region that separates the 3' splice acceptor site from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

10. The nucleic acid molecule of claim 9 wherein the 3' splice region further comprises a branch point and a pyrimidine tract.

11. A nucleic acid molecule comprising: (a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within a cell; (b) a 5' splice site; (c) a spacer region that separates the 5' splice site from the target binding domain; and (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

12. The nucleic acid molecule of claim 9 or 10 wherein the nucleic acid molecule further comprises a 5' donor site.

13. A method for targeting cell death comprising: (i) contacting said cell with a nucleic acid molecule wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 3' region comprising a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence encoding a light producing protein enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell; and (ii) placing a photosensitizer in close enough proximity to the cell to permit activation of the photosensitizer by the light producing enzyme, wherein said activation results in cell death.

14. The method of claim 13 wherein said 3' splice region further comprises a branch point and a pyrimidine tract.

15. A method for targeting cell death comprising: (i) contacting said cell with a nucleic acid molecule wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 5' splice site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence encoding a light producing protein enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell; and (ii) placing a photosensitizer in close enough proximity to the cell to permit activation of the photosensitizer by the light producing enzyme, wherein said activation results in cell death.

16. The method of claim 13 or 14 wherein the nucleic acid molecule further comprises a 5' donor site.

17. The method of claim 13, 14 or 15 further comprising contacting said cell with a substrate specific for the light producing protein or enzyme.

18. The method of claim 16 further comprising contacting said cell with a substrate specific for the light producing protein or enzyme.

19. A recombinant conditionally replicative adenovirus comprising a transgene wherein said transgene encodes one or more pre-trans-splicing molecules wherein said pre-trans-splicing molecules comprise: a) one or more target binding domains that target binding of the pre-trans-splicing molecule to a target pre-mRNA expressed within a cell; b) a 3' splice region comprising a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a polypeptide selected from the group consisting of (i) an adenovirus polypeptide; or (ii) a polypeptide that functions as a light inducing enzyme or protein.

20. The recombinant conditionally replicative adenovirus of claim 19 wherein said 3' splice region further comprises a branch point and a polypyrimidine tract.

21. A recombinant conditionally replicative adenovirus comprising a transgene wherein said transgene encodes one or pre-trans-splicing molecules wherein said pre-trans-splicing molecules comprise: a) one or more target binding domains that target binding of the pre-trans-splicing molecule to a target pre-mRNA expressed within a cell; b) a 5' splice site; c) a spacer region that separates the 5' splice site from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a polypeptide selected from the group consisting of (i) an adenovirus polypeptide; or (ii) a polypeptide that functions as a light inducing enzyme or protein.

22. The adenovirus of claim 20 or 21 wherein the pre-trans-splicing molecule further comprises a 5' donor site.

23. A recombinant conditionally replicative adenovirus comprising (i) a transgene wherein said transgene encodes a pre-trans-splicing molecules wherein said pre-trans-splicing molecules comprises a) one or more target binding domains that target binding of the pre-trans-splicing molecule to a target pre-mRNA expressed within a cell; b) a 3' splice region comprising a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an adenovirus polypeptide; and (ii) a transgene encoding a light producing protein or enzyme.

24. The recombinant conditionally replicative adenovirus of claim 23 wherein said 3' splice region further comprises a branch point and a polypyrimidine tract.

25. A recombinant conditionally replicative adenovirus comprising (i) a transgene wherein said transgene encodes a pre-trans-splicing molecule wherein said pre-trans-splicing molecule comprises: a) one or more target binding domains that target binding of the pre-trans-splicing molecule to a target pre-mRNA expressed within a cell; b) a 5' splice site; c) a spacer region that separates the 5' splice site from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an adenovirus polypeptide; and (ii) a transgene encoding a light producing enzyme or protein.

26. The adenovirus of claim 23 or 24 wherein the pre-trans-splicing molecule further comprises a 5' donor site.

27. A method for targeting cell death comprising contacting said cell with the conditionally replicative adenovirus capable of encoding a light producing enzyme or protein.

maxwellsdemon - 1 Mar'05 - 15:54 - 7944 of 23903 (premium)


2. BACKGROUND OF THE INVENTION

[0003] 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.

[0004] The mechanism of spliced leader trans-splicing, which is nearly identical to that of conventional cis-splicing, proceeds via two phosphoryl transfer reactions. The first causes the formation of a 2'-5' phosphodiester bond producing a `Y` shaped branched intermediate, equivalent to the lariat intermediate in cis-splicing. The second reaction, exon ligation, proceeds as in conventional cis-splicing. In addition, sequences at the 3' splice site and some of the snRNPs which catalyze the trans-splicing reaction, closely resemble their counterparts involved in cis-splicing.

[0005] Trans-splicing may also refer to a different process, where an intron of one pre-mRNA interacts with an intron of a second pre-mRNA, enhancing the recombination of splice sites between two conventional pre-mRNAs. This type of trans-splicing was postulated to account for transcripts encoding a human immunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et al., 1989, Proc. Nat'l. Acad. Sci. USA 86:8020). In addition, trans-splicing of c-myb pre-RNA has been demonstrated (Vellard, M. et al. Proc. Nat'l. Acad. Sci., 1992 89:2511-2515), trans-spliced RNA transcripts from SV40 have been detected in cultured cells and nuclear extracts (Eul et al., 1995, EMBO. J. 14:3226) and more recently, the transcript from the p450 gene in human liver has been shown to be trans-spliced (Finta et al., 2002, J. Biol Chem 22:5882-5890). However, in general, naturally occurring trans-splicing of mammalian pre-mRNAs is thought to be an exceedingly rare event.

[0006] In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several groups (Konarska & Sharp, 1985, Cell 46:165-171 Solnick, 1985, Cell 42:157; Chiara & Reed, 1995, Nature 375:510). Reasonably efficient trans-splicing (30% of cis-spliced analog) was achieved between RNAs capable of base pairing to each other, splicing of RNAs not tethered by base pairing was further diminished by a factor of 10. Other in vitro trans-splicing reactions not requiring obvious RNA-RNA interactions among the substrates were observed by Chiara & Reed (1995, Nature 375:510), Bruzik J. P. & Maniatis, T. (1992, Nature 360:692) and Bruzik J. P. and Maniatis, T., (1995, Proc. Nat'l. Acad. Sci. USA 92:7056-7059). These reactions occur at relatively low frequencies and require specialized elements, such as a downstream 5' splice site or exonic splicing enhancers.

[0007] U.S. Pat. Nos. 6,083,702, 6,013,487 and 6,280,978 describe the use of PTMs to mediate a trans-splicing reaction by contacting a target precursor mRNA to generate novel chimeric mRNAs. The resulting RNA can encode any gene product including a protein of therapeutic value to the cell or host organism, a toxin, such as Diptheria toxin subunit A, which causes killing of the specific cells or a novel protein not normally present in cells. The PTMs can also be engineered for the production of chimeric proteins including those encoding reporter molecules useful to image gene expression in vivo in real time or to add peptide affinity purification tags which can be used to purify and identify proteins expressed in a specific cell type.

[0008] Photodynamic therapy (PDT) of cancer uses light excitation of a photosensitive substance to produce oxygen-related cytotoxic intermediates, such as singlet oxygen or free radicals (Dougherty et al., 1993, Photochem. Photobiol. 58:895-900; Hopper et al., 2000, Lancet Oncol. 1:212-219; Ochsner et al., 1997, J. Photochem. Photobiol. B. Biol 39:1-18, Fuchs et al., 1998, Biol. Med. 24:835-847). For example, the use of CL.sup.4 for the excitation of the photosensitizer hypercin has been used for the in vitro inactivation of the equine infectious anemia virus (Carpenter, S. et al. 1994, Proc. Natl. Acad. Sci. USA 91:12273-12277). Additionally, Theodossis et al., described the in vitro photodynamic effect of rose bengal activated by intracellular generation of light generated by the oxidation of the chemiluminescent substrate luciferin, in luciferase-transfected NIH 3T3 murine fibroblasts (Theodossis et al., 2003, Cancer Research 63:1818-1821).

[0009] PDT involves the use of two individual components that combine to induce cytotoxicity in an oxygen dependent manner. The first component of PDT is a photosensitizer molecule that usually enters cells and/or tissues non-specifically. The second component involves the localized administration of light of a specific wavelength that is capable of activating the photosensitizer. Once activated the photosensitizer transfers energy from the light to molecular oxygen, thereby generating reactive oxygen species (ROS), such as singlet oxygen and free radicals. Such ROS mediate cellular toxicity. Photosensitizers may also undergo photochemical reactions that do not use oxygen as an intermediate, such as compounds that result in photoaddition to DNA. As used herein, the term photosensitizer includes, but is not limited to, other chemicals that are activated upon exposure to light. Such photosensitizers are known to those skilled in the art and the examples set forth herein are non-limiting.

[0010] Although photodynamic therapy use is desirable because of its limited side effects, its main disadvantages are the poor accessibility of light to certain tissues and the problem of restricting the delivery of light primarily to the target cells. The present invention provides methods and compositions for targeted expression of light producing enzymes in the desired cell types and in cells that otherwise are inaccessible to light, thereby providing a method for use of photodynamic therapy for the specific destruction of targeted cells. Specifically, the invention provides PTM molecules that are designed to interact with one or more cell selective target pre-mRNA species and mediate trans-splicing reactions resulting in the generation of chimeric mRNA molecules capable of encoding light producing enzyme or protein. The expression of the light producing enzyme or protein permits activation of a co-localized photosensitizer leading to death of the selected cell. The present invention provides a system for targeting cancer cell destruction. In addition, the invention provides a system for targeting selective cell death to cells infected with pathogenic microorganisms, or, cell death in instances where the activity of a particular cell type leads to disease.

3. SUMMARY OF THE INVENTION

[0011] The present invention provides methods and compositions for conferring selective death on cells expressing a specific target precursor messenger RNA (selective target pre-mRNAs). The compositions of the invention include 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 mRNA molecules (chimeric mRNA) capable of encoding light producing proteins or enzymes. Light producing proteins include those molecules capable of photoactivating a photosensitizer sufficient to result in formation of cytotoxic intermediates, including cytotoxic oxygen related-intermediates. Light producing proteins include those capable of fluorescence, FRET (fluorescent resonance energy transfer), and phosphorescence. Upon successful trans-splicing between the target pre-mRNA and the PTM, the light producing proteins or enzymes are expressed thereby providing the activity necessary for activation of the photosensitizer. Such activation leads to cell death, thereby targeting selective destruction of specific cells (FIG. 1A).

[0012] The present invention provides methods and compositions for conferring selective death on cancer cells expressing specific target precursor messenger RNA molecules (cancer cell selective target pre-mRNAs). The compositions of the invention PTMs are designed to interact with one or more cancer cell selective target pre-mRNA and mediate trans-splicing reactions resulting in the generation of novel chimeric mRNA molecules (chimeric mRNA) capable of encoding a light producing protein or enzyme. The portion of the target pre-mRNA trans-spliced to the PTM provides the signal sequences necessary for translation of the chimeric mRNA molecule. The portion of the PTM trans-spliced to the target pre-mRNA provides sequences encoding light producing enzymes that provide essential activity necessary for activation of cytotoxic photo sensitizers.

[0013] The methods and compositions of the invention provide a means for selective destruction of cancer cells within a tumor. Since the viability of tumor cells relies on the supply of nutrients via the bloodstream, targeting of cells of the vascular system may also be used to treat cancer. In such instances, the selective target pre-mRNA is a pre-mRNA expressed in the cells in newly created regions of the vascular system. Thus, the present invention provides methods and compositions for treating a variety of different cancers including but not limited to, breast, prostate, bladder, pancreatic or liver cancer.

[0014] In addition the present invention provides methods and compositions for conferring selective death on cells expressing mRNAs produced by a pathogenic infectious agent. In such instances the PTM is designed to interact with one or more target pre-mRNAs produced by the pathogenic infective agent. The portion of the target pre-mRNA produced by, or in response to, the pathogen and trans-spliced to the PTM provides the signal sequences necessary for translation of the chimeric molecule. The portion of the PTM trans-spliced to the target pre-mRNA provides sequences encoding the light producing proteins or enzymes that provide an essential activity necessary for activation of a cytotoxic photosensitizer. The methods and compositions of the invention may be utilized for selective destruction of infected cells.

[0015] In yet another embodiment of the invention, the methods and compositions of the invention may be used for conferring cell death in a subject where the activity of that cell leads to a disease state, for example, an immune or hormonal disorder.

maxwellsdemon - 1 Mar'05 - 15:58 - 7945 of 23903 (premium)


Presumably the patient is going to have to hide in a dark room?? :-)

small crow - 1 Mar'05 - 21:47 - 7955 of 23903


md

Fantastic find - thankyou. Intronn has no income yet but enormous ingenuity and, again, this is potentially huge. The thing that is especially fascinating to me, but where I need the advice of a physicist .... is in the possiblity of combining tumour imaging with tumour death by these methods. For example, could the FRET mode be used to image the tumour before zapping by does of the photosensitive toxin, and then be used to image the reduction of the tumour in response?


maxwellsdemon - 2 Mar'05 - 15:41 - 7978 of 23904 (premium)


small crow,

I see you are the only person to pick up on the ideas described in the patent application.

On your point regarding imaging:As a safety measure, I would think that the it would be possible to first check that the light emission was not occuring in observable places other than those affected by cancer prior to the application of the photoactivated toxin. I suppose it could also be used to image the cancer itself, as you suggest, but the applications suggested in patent are for cancers which cannot be illuminated externally. So for these cases,with the cancer occuring deep within the body, it is difficult to see how it could be imaged.

small crow - 2 Mar'05 - 16:05 - 7979 of 23904


md

I am physically useless but no chance of using something analogous to tomography if some kind of resonance is involved? There, ignorance demonstrated. If so, could be very interesting - real time observation of recession.

DoobyDave - 2 Mar'05 - 16:18 - 7980 of 23904 (premium)


Small crow,

You say 'real time recession'. How much time did you have in mind? Cell apoptosis usually takes hours, culminating in cell wall rupture. I can't imagine a scan imaging that in anything but experimental conditions.

I would also have thought that any imaging associated with the patented technique would have to use a quite different wavelength to that of light, for the reasons that maxwellsdemon explains.


DD

That's enough reading for one day.