Gene Silencing
The biggest threat to long-term survival of
cancer patients is multi-drug resistance.
After treatment with conventional drug therapy, a large number of cancer cells die but a small percentage of the cells can produce proteins that make cancer cells resistant to chemotherapeutic drugs. Consequently, follow-up drug treatments can fail. The tumours thus become untreatable and continue to flourish, ultimately killing the patient.
The genes of cancer cells readily mutate to over-produce multi-drug resistance proteins that can make tumours totally resistant to conventional chemotherapeutic treatments. The starting point for the treatment of these drug-resistant cancers came from the current very active area of worldwide research which has shown that small interfering RNAs (siRNAs) can silence disease-causing genes.
EnGeneIC’s initial breakthrough was the discovery that EDVs could readily be packaged with therapeutically significant concentrations of siRNA. These siRNAs can be designed to silence multi drug resistance proteins or any protein that can lead to cancer cell progression or resistance to apoptosis. When these siRNA-packaged, antibody-targeted EDVs were delivered into the blood system, they quickly dropped out of the leaky blood vessels that normally surround the tumour mass. Then, because the EDVs are armed with targeting antibodies, they then lock on to the surface antigens of the cancer cells, enter into the cancer cells and release the siRNA. The siRNA is highly effective in switching off the production of the multi-drug resistance protein and the same cancer cells were now exquisitely sensitive to tiny concentrations of chemotherapeutic drugs.
A further breakthrough occurred when it was shown that the dual sequential treatment of siRNA-packaged EDVs followed by drug-packaged EDVs could effectively treat a range of different human cancers that were implanted in mice. In other words, the drug resistant cancer cells could be transformed into being drug sensitive, and targeted for cell death (SEE FIGURE2). This reversal of drug resistance followed by chemotherapy was extremely effective even in highly aggressive cancers and resulted in 100% survival in mice that received the dual sequential treatment.
Testing over two years achieved highly significant tumour regression/stabilisation in the experimental groups by using substantially lower concentrations of drug and antibodies than used in conventional treatments. Similar success has been achieved in early-stage clinical case studies in dogs suffering from relapsed drug-resistant cancers.
Concept of dual sequential therapy (nanocell-mediated delivery
of siRNA followed by drug) to treat drug resistant cancers. In a
single figure it shows EDVs emerging from the tumor associated
leaky vasculature and entering into a tumor microenvironment.
A single tumor cell is depicted which in clockwise segments
undergoes the sequential therapy to result in tumor cell death
in the last segment.
In brief (clockwise shown by purple arrow):
Segment 1: tumor cells over-expressing, as an example,
multi-drug resistance mediating protein (purple) hence
preventing drug (red) entry into the cell.
Segment 2: tumor receptor-targeted, siRNA-packaged
minicells
docking onto tumor cell surface receptor (green).
Segment 3: EDVs endocytosed and degraded in
intracellular
lysosomes, releasing siRNA into the cytoplasm.
Segment 4: siRNA strands separate and bind to target mRNA (yellow).
Segment 5: Target mRNA cleavage and cell surface showing
knockdown of the multi-drug resistance mediating protein
(disappearing purple protein).
Segment 6: Tumor receptor-targeted, drug-packaged minicells
docking onto the tumor cell surface receptor (green).
Segment 7: EDVs are endocytosed, degraded in lysosomes
and releasing the drug into the cytoplasm and nucleus.
Segment 8: Tumor cell is flooded with the drug
and the cell crumbles and dies.
The above figure is published in Nature Biotechnology, July 2009.
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Additional advantages of siRNA
and shRNA packaging in EDVs:
• A single EDV can package ~12,000 siRNA molecules.
• Multiple siRNAs of different types can be packaged into EDVs.
• EDVs can carry therapeutically significant concentrations of shRNA
encoding plasmids, in which a single EDV can carry ~100 plasmid copies. |