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01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

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Excessive necrotic death in cells of the coronary artery<br />

wall results by oxygen and glucose deprivation after<br />

blockage of a blood vessel feeding a segment of the heart<br />

(also the brain in stroke). Destructive free radicals are then<br />

produced during inflammation of the area which can cause<br />

apoptotic or necrotic death in cells in the surroundings.<br />

Since both brain and heart cells in the adult are not<br />

regenerated, Biotech Companies (for example <strong>Gene</strong>ntech)<br />

are focusing in developing drugs that block free radical<br />

formation, inhibit ICE-like proteases, or inhibit apoptosis<br />

via other mechanisms.<br />

The progressive loss of neuron cells in senile or other<br />

brain diseases such as Alzheimer’s disease, Parkinson’s<br />

disease, Huntington’s disease, and amyotrophical lateral<br />

sclerosis may ensue by apoptosis. Etiologic factors may<br />

include excessive levels of neurotransmitters, low levels of<br />

NGF, free radical-mediated damage, and deregulation in<br />

the expression of genes encoding apoptotic regulators<br />

during aging. Deregulation in apoptosis may also have a<br />

share in the induction of osteoporosis.<br />

XXI. <strong>Gene</strong>s involved in the regulation of<br />

apoptosis as targets for gene <strong>therapy</strong><br />

Many of the molecular controllers of apoptosis<br />

including cytokine signaling pathways (TNF-α, IL-1β),<br />

tumor suppressor proteins (p53), viral proteins (E1A of<br />

adenovirus), cellular oncoproteins (Myc), proteins that<br />

control the cell cycle (E2F), apoptosis inducers (Bax) and<br />

antiapoptotic molecules (Bcl-2, NF-κB) could constitute<br />

potential targets for pharmacological intervention for the<br />

treatment not only of cancer but of other human disease.<br />

Although for cancer treatment it is desirable to induce<br />

apoptosis, the opposite effect, that is inhibition of<br />

apoptotic pathways is desirable in the gene <strong>therapy</strong> of<br />

heart disease and degenerative brain disease (see below).<br />

A. <strong>Gene</strong> <strong>therapy</strong> that targets bcl-2<br />

Bcl-2 protein is overexpressed in a variety of human<br />

leukemias because of translocation of its gene to the<br />

immunoglobulin locus; Bcl-2 is associated with the outer<br />

surface of the mitochondrion and appears to be involved in<br />

scavenging oxygen radicals. Overexpression of the bcl-2<br />

gene in tumors is thought to be responsible for the poor<br />

response of the tumors to antineoplastic drugs and<br />

radiation <strong>therapy</strong> blocking apoptosis of the tumor cells.<br />

Bcl-2 can interact with members of the Bcl-2 family<br />

including Bax, Bcl-X-S, Bcl-X-L, and Mcl-1 but also with<br />

heterologous protein molecules including BAG-1, Raf-1,<br />

and R-Ras.<br />

Introduction of the bcl-2 gene into human diploid<br />

breast epithelial MCF10A cells (containing the wild-type<br />

p53 gene) resulted in suppression in p21 gene expression<br />

although the level of expression of p53 was not affected;<br />

<strong>Gene</strong> Therapy and <strong>Molecular</strong> <strong>Biology</strong> Vol 1, page 69<br />

69<br />

these studies suggested that Bcl-2 may inhibit the<br />

functional activity of p53 protein and might regulate the<br />

commitment of cells to commit suicide or proliferate<br />

(Upadhyay et al, 1995).<br />

Overexpression of the bcl-2 gene in tumors is thought<br />

to be responsible for the poor response of the tumors to<br />

antineoplastic drugs and radiation <strong>therapy</strong> blocking<br />

apoptosis of the tumor cells; therefore, down-regulation of<br />

the bcl-2 gene specifically in tumor cells could induce<br />

apoptosis. Primary untreated human prostate cancers were<br />

found to express significant levels of this apoptosissuppressing<br />

oncoprotein; this is a striking difference with<br />

normal prostate secretory epithelial tissue not expressing<br />

Bcl-2 (Raffo et al, 1995). Transfection of LNCaP human<br />

prostate cancer cells with a plasmid expressing bcl-2<br />

rendered these cells highly resistant to a variety of<br />

apoptotic stimuli (serum starvation or treatment with<br />

phorbol ester) and induced earlier and larger tumors in<br />

nude mice. The ability of Bcl-2 to protect prostate cancer<br />

cells from apoptotic stimuli correlated with the ability of<br />

the cells to form hormone-refractory prostate tumors in<br />

nude mice (Raffo et al, 1995).<br />

The Bcl-2 oncoprotein suppresses apoptosis and, when<br />

overexpressed in prostate cancer cells, makes these cells<br />

resistant to a variety of therapeutic agents, including<br />

hormonal ablation. Overexpression of BCL-2 is common<br />

in non-Hodgkin lymphoma leading to resistance to<br />

apoptosis and promoting tumorigenesis. Therefore, bcl-2<br />

provides a strategic target for the development of gene<br />

knockout therapies to treat human prostate cancers (Dorai<br />

et al, 1997) and non-Hodgkin lymphomas (Webb et al,<br />

1997).<br />

Down-regulation of Bcl-2 can be accomplished with<br />

antisense. In patients with relapsing non-Hodgkin<br />

lymphoma, BCL-2 antisense <strong>therapy</strong> led to an<br />

improvement in symptoms; antisense oligonucleotides<br />

targeted at the open reading frame of the BCL-2 mRNA<br />

showed effectiveness against lymphoma grown in<br />

laboratory animals and has entered human clinical trials.<br />

The first study was conducted on nine patients with BCL-<br />

2-positive relapsed non-Hodgkin lymphoma using a daily<br />

subcutaneous infusion of 18-base, fully phosporothioated<br />

antisense oligonucleotide administered for 2 weeks (Webb<br />

et al, 1997). A local inflammation at the infusion site was<br />

noted. A reduction in tumour size was observed in two<br />

patients (one minor, one complete response) using<br />

computed tomography scans; in two other patients, the<br />

number of circulating lymphoma cells decreased during<br />

treatment. In four patients, serum concentrations of lactate<br />

dehydrogenase fell, and in two of these patients symptoms<br />

improved (Webb et al, 1997).<br />

A divalent hammerhead ribozyme, constructed by<br />

recombining two catalytic RNA domains into an antisense<br />

segment of the coding region for human bcl-2 mRNA was<br />

able to rapidly degrade bcl-2 mRNA in vitro; it was then

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