Strategies for short hairpin RNA delivery in cancer gene therapy

6002 Background: Human papillomavirus type 16 is identified in almost 50% of the cases of OSCC. The E6 and E7 genes of HPVs encode oncoproteins that bind and degrade the tumor suppressor proteins p53 and Rb, respectively. We are exploring the potential use of short hairpin RNA (sh RNA) for gene therapy of HPV-positive OSCC. Methods: Small hairpin RNAs targeting E6 or E7 genes were delivered by a retrovirus vector to 93VU147T (bearing integrated HPV16 DNA) and 92VU040T (HPV negative) oropharyngeal cancer cell lines. Flow cytometry analysis was used to assess apoptosis after the retrovirus infection. The E6 and E7 mRNA downregulation was assessed by reverse transcription polymerase chain reaction (RT-PCR). At protein level p53 and Rb expression were evaluated with Western blotting analysis. Results: Apoptosis was seen in over 90% of 93VU147T cells 48 hours after infection whereas 92VU040T cells were not affected. RT-PCR demonstrated that HPV16 E6/E7 mRNA levels decreased significantly in infected 93VU147T cells. 93VU147T infected cells also showed a marked increase in p53 and Rb protein levels. Conclusions: Downregulation of E6/E7 gene expression in HPV16+ OSCC cells results in apoptosis and reactivation of p53 and Rb tumor suppression pathways. These results have significant implications in treating HPV-associated OSCC with HPV-targeted gene therapy. No significant financial relationships to disclose.

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685: A Short Hairpin RNA Targeting Pin (Protein Inhibitor of NOS) is More Efficient than the Anti-Sense Pin RNA for Gene Therapy of Aging-Related Erectile Dysfunction in the RAT

The Journal of Urology ◽

10.1016/s0022-5347(18)30925-x ◽

2007 ◽

Vol 177 (4S) ◽

pp. 230-230

Author(s):

Thomas R. Magee ◽

Istvan Kovanecz ◽

Liliana Cantini ◽

Monica G. Ferrini ◽

Dolores Vernet ◽

...

Keyword(s):

Gene Therapy ◽

Erectile Dysfunction ◽

Short Hairpin Rna ◽

Protein Inhibitor ◽

Hairpin Rna ◽

Rna Targeting ◽

Short Hairpin

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Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052320 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2320

Author(s):

Made Angga Akwiditya ◽

Chean Yeah Yong ◽

Mohd Termizi Yusof ◽

Abdul Razak Mariatulqabtiah ◽

Kok Lian Ho ◽

...

Keyword(s):

Gene Therapy ◽

Hepatitis B ◽

Cancer Cells ◽

Targeted Delivery ◽

Short Hairpin Rna ◽

Core Antigen ◽

Hairpin Rna ◽

Post Transfection ◽

Virus Like Particle ◽

Short Hairpin

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells’ viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

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