Marked for Death

Using Magic Targets, not Magic Bullets, to treat cancer

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Proposal #1: Marked for Death

Proposal #2: Centralized Proposal Depository 

Proposal #3: Inducible tract-specific spinal cord injury

Suggestion #1: Take the 'preclinical' out of animal testing 

Suggestion #2: Public Contribution Rating

Suggestion #3: Crowdsource CRISP

Proposals Under Construction

THE WIKI VERSION OF THIS PROPOSAL IS POSTED HERE

PLEASE MAKE ANY COMMENTS, SUGGESTIONS, MODIFICATIONS, OR ADDITIONS YOU WANT AT THAT LINK!

 

Submitted 12/30/2007 by Noam Y. Harel 

 

HYPOTHESIS

Cancer-prone tissues can be prophylactically transduced with a library of lentiviral constructs encoding a death gene under control of unique siRNA-targeting sequences. By sequencing DNA from subsequently arising tumors, matched siRNAs may be used to induce death gene expression. The death gene will allow selective killing of all cells clonally derived from the cell containing the targeted genetic tag. This system provides a potent weapon against clonally derived cancers.


RATIONALE/BACKGROUND

Cancers can and will arise from any cell in the body. In most cases, tumors derive from a clonal cell population. In a few cases, tumors express distinct markers, such as the chromosomal translocation leading to the bcr-abl fusion kinase in chronic myelogenous leukemia (Daley et al., 1990; Nowell and Hungerford, 1960). Such tumor-specific tags, when identifiable, provide a target for so-called ‘magic bullet’ anti-cancer drugs – imatinib, for instance (Druker et al., 2001).

But the great majority of tumors do not express known targets for magic bullets. To generate these targets, is there a way to tag cells with unique genetic codes before they become cancers? What if these unique codes were used to control expression of an inducible death gene that would allow selective killing of all tumor cells clonally derived from cells with that code? This proposal outlines a feasible approach to these questions, integrating several key technologies: RNA interference, bioinformatics, and lentiviral gene therapy.

Inducible expression with RNA interference

Traditional inducible expression constructs rely on drugs such as doxycycline or tamoxifen to mediate increased transcription of the gene of interest (Goverdhana et al., 2005; Szulc et al., 2006). At the desired time, one drug induces gene expression in all cells transduced with the construct. But what if we desire to stimulate expression in only a subset of the transduced cells? This feature requires a mechanism more complex than traditional drug-induced constructs.

The crux of the RIDD (RNAi-Inducible Death Dimer) system to be used in this proposal is the ability to use customizable short interfering RNAs (siRNAs) to induce gene expression only in targeted cells. siRNAs induce the endogenous RNA interference (RNAi) pathway, leading to degradation of mRNAs that harbor complementary sequences to the siRNA (Elbashir et al., 2001). In the RIDD system, degradation of the repressor mRNA will lead to induction of the death dimer. By varying the sequence at the siRNA-targeting site, a library of RIDD constructs may be composed, each selectively inducible by the appropriately matched siRNA. At the desired time (for instance, when a RIDD-transduced cell becomes cancerous), the death gene may be specifically induced in the responsible cell and its progeny, allowing selective killing of tumor cells.

Bioinformatics to optimize a library of unique siRNA-targeting sites

Sequences composing the siRNA-targeting sites that regulate the RIDD system need to satisfy two requirements: 1) mediate effective knockdown of the targeted RNA; 2) avoid similarity with endogenous sequences that could lead to harmful off-target effects. Genomic bioinformatics approaches can drastically reduce the empiric testing that would be required to meet these requirements. Freely available online siRNA optimization tools will help with the first requirement. To meet the second requirement, part of each targeting sequence will consist of an 11 nucleotide “nullomer” that is absent from the human genome (Acquisti et al., 2007; Hampikian and Andersen, 2007). 

Lentiviral gene therapy

The Magic Target approach necessitates insertion of a genetic construct into a cell’s genome such that the DNA is passed on to all of that cell’s progeny. Retroviral vectors provide the most efficient method for mediating integration of exogenous DNA into host chromosomes (Thomas et al., 2003). Lentiviruses comprise a class of retroviruses that are able to integrate into the chromosomes of non-dividing cells as well as dividing cells. Even though this proposal plans on tagging cells that may become cancerous (i.e. dividing out of control), the tags will be administered one or more times before cells become cancerous, at times when they may not be dividing. As with other types of retroviral gene therapy vectors, many measures have been taken to ensure that lentiviral vectors have almost no risk of pathogenicity (Thomas et al., 2003).

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