RNA Multifunctional Antisense Gene Transfer Strategy to Alter Hepatocyte Growth Factor Receptor Expression in Glioblastoma Multiforme

• Published in: The FASEB journal Vol. 30; no. S1
• Main Author: Patel, Priyal
• Format: Journal Article
• Language: English
• Published: 01-04-2016
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.30.1_supplement.590.6

Abstract only
The most common central nervous system malignancy is glioblastoma multiforme (GBM). The aggressive tumor spreads rapidly migrating through the white matter of the brain. Current therapy includes surgical removal, radiation and chemotherapy extending survival 14 months. Furthermore, the blood‐brain barrier is a challenge to targeted therapy. Our research is to develop a novel gene transfer vector to deliver the DNA encoding an antisense RNA (ASR) to alter the splicing pattern of the hepatocyte growth factor receptor (HGFR). In this approach, the ASR blocks recognition of splice sites and splicing enhancers. The current strategy targets the splicing of exons 11 to 12 of the pre‐mRNA transcript. Blockage of this splicing event leads to retention of intron 11. Because this intron includes 14 intronic alternative polyadenylation (IPA) signals, there is great potential to generate a shortened transcript and its subsequent soluble therapeutic HGFR decoy. A 1
st
generation construct containing an antisense with a human U7 snRNA was transfected into human glioblastoma cell lines, A172 and U87MG. The 1
st
generation therapy was successfully expressed in transfected cell lines. To optimize therapeutic efficiency, we have designed a 2
nd
generation gene transfer construct with multiple elements with distinct functions. These include the ASR with an hnRNPA1 splicing inhibitory element and an sm‐Opt U7snRNA (sm‐Opt/U7) localization signal with the novel tertiary ‘kiss domain’. The ASR targets the 5′ splice site of Exon 11, the sm‐Opt/U7 signal stabilizes and directs the ASR into the spliceosome of the nucleus, the hnRNPA1 tail blocks the splicing machinery and the ‘kiss domain’ stabilizes tertiary structure of the therapy vector. The new design makes use of the U7 promoter and 3′ transcription termination element to allow more efficient transcription. Using the 1
st
generation therapy vector we have developed assays to measure efficiency of therapy vector delivery, as well as levels of pre‐mRNA and spliced HGFR transcript. In the next steps, we are delivering the 2
nd
generation therapy vector to measure the efficacy to increase the abundance of shortened transcript and reduction of full length HGFR in the GBM cells. Real‐ time polymerase chain reaction (PCR) and ELISA will be used to determine the ratios of the pre‐mRNA, mRNA and decoy in the cell and in the media.