Gene Therapy - Arrigo De Benedetti, Ph.D.

Key Member

Arrigo De Benedetti, Ph.D.
Dept. of Biochemistry

See the Curriculum Vitae
Benedetti Lab

     Cancer gene therapy is at the forefront of medicine, but it is currently subject to three major constraints:

     (1) method of delivery,
     (2) specific gene expression, and
     (3) efficacy vs. toxicity.

One promising strategy of cancer gene therapy is based on the herpes thymidine kinase (HTK)/ganciclovir (GCV) system. Most of the work has been directed at targeting primary tumors, whereas treatment of metastasis requires methods of specific delivery. We propose a novel approach targeting a characteristic that distinguishes cancer from normal cells, i.e., elevated eukaryotic translation initiation factor (eIF) 4E, and ultimately allowing for selective killing. eIF4E is a component of the helicase that unwinds excess structure in the 5'UTR of mRNAs. Elevated eIF4E specifically facilitates the translation of mRNAs with a long and structured 5'UTR. With this in mind, the expression of HTK was selectively regulated by placing the 5'UTR of FGF2, previously found to be translationally regulated, upstream of the HTK open reading frame. The idea behind this construct is to obtain a more selective target to GCV killing by limiting the expression of HTK to cancer cells while sparing the population of normal cells, which are unable translate this mRNA. This is important, because in addition to selectively kill cancer cells at the primary tumor site, the ability to selectively attack metastasis could be possible. Preliminary results show that this was feasible in one cell model. Experiments are proposed with a panel of normal and cancer cell lines to determine the general applicability of the system: pattern of HTK expression, differential sensitivity to GCV, and enzymatic activity. Additional constructs with synthetic hairpins at 5'UTR will also be tested. Modulation of HTK mRNA level by induction of the vector promoter, in relation to translational regulation will be established. Lipo-transfection of some of these constructs in mice will follow to determine efficacy (i.e., tumor regression) vs. toxicity to distal organs. The capacity to reduce or eliminate lung metastases with low toxicity and to extend the survival of mice will be tested. All these effects will be correlated with the distribution of the vectors in tumors and normal tissues by real-time PCR analysis and IHC. The pattern of expression of HTK will also be monitored by a variety of methods, including an in vivo imaging (PET) system that takes advantage of the conversion and incorporation of [18F]GCV by cells expressing HTK. This allows for direct monitoring of the pattern of HTK expression in tumors and normal organs without killing the animals, which can be subsequently treated with GCV to eliminate the tumor nodules. This will confirm the actual regression of metastases by the modified vectors, and correlate the possible cytopathic effects with the expression of HTK in different organs.

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