Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells
Approximately 80%–90% of uveal melanomas (UVM) harbor a single base pair substitution in one of two Gα protein subunits (GNAQQ209L/P/GNA11Q209L), resulting in constitutive activation and tumor initiation/progression. Herein, a small interfering RNA (siRNA) that specifically targets GNAQQ209L transcr...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
|
| Series: | Molecular Therapy: Oncology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S295032992500089X |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849248155571322880 |
|---|---|
| author | Trace F. McCall Emma J. Sawyer Joshua Darnell Matthew L. Hirsch Jacquelyn J. Bower |
| author_facet | Trace F. McCall Emma J. Sawyer Joshua Darnell Matthew L. Hirsch Jacquelyn J. Bower |
| author_sort | Trace F. McCall |
| collection | DOAJ |
| description | Approximately 80%–90% of uveal melanomas (UVM) harbor a single base pair substitution in one of two Gα protein subunits (GNAQQ209L/P/GNA11Q209L), resulting in constitutive activation and tumor initiation/progression. Herein, a small interfering RNA (siRNA) that specifically targets GNAQQ209L transcripts induced significant cell death in GNAQQ209L UVM cells, whereas little to no effects were observed on GNAQwt cells or GNAQwt transcripts. The most effective siRNA sequence was subsequently encoded into a short hairpin RNA (shRNA) cassette (shGNAQQ209L), expressed in a recombinant adeno-associated virus (rAAV), and the AAV2 capsid was selected for viral production upon completion of a serotype survey in UVM cells. Transduction with rAAV2-shGNAQQ209L induced significant cell death in GNAQQ209L cells but not in a GNAQwt UVM line. Unexpectedly, cell death in the GNAQQ209L UVM cells was also observed upon transduction with the non-targeting control rAAV2 (although to a lesser degree than rAAV2-shGNAQQ209L), suggesting that an element of the AAV vector itself exhibits toxicity in GNAQQ209L UVM cells. This work is among the first describing a genetic-based rAAV approach to specifically target an oncogenic mutant driver allele using single base pair allelic discrimination, collectively demonstrating that both siRNA and rAAV methods of GNAQQ209L depletion result in significant UVM cell death. |
| format | Article |
| id | doaj-art-172098a1fe9740eca92cf6c6387c1663 |
| institution | Kabale University |
| issn | 2950-3299 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Oncology |
| spelling | doaj-art-172098a1fe9740eca92cf6c6387c16632025-08-20T03:58:00ZengElsevierMolecular Therapy: Oncology2950-32992025-09-0133320102010.1016/j.omton.2025.201020Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cellsTrace F. McCall0Emma J. Sawyer1Joshua Darnell2Matthew L. Hirsch3Jacquelyn J. Bower4Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Eye Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USADepartment of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Eye Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USADepartment of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Eye Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USADepartment of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Eye Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USADepartment of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Eye Research Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Corresponding author: Jacquelyn J. Bower, Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.Approximately 80%–90% of uveal melanomas (UVM) harbor a single base pair substitution in one of two Gα protein subunits (GNAQQ209L/P/GNA11Q209L), resulting in constitutive activation and tumor initiation/progression. Herein, a small interfering RNA (siRNA) that specifically targets GNAQQ209L transcripts induced significant cell death in GNAQQ209L UVM cells, whereas little to no effects were observed on GNAQwt cells or GNAQwt transcripts. The most effective siRNA sequence was subsequently encoded into a short hairpin RNA (shRNA) cassette (shGNAQQ209L), expressed in a recombinant adeno-associated virus (rAAV), and the AAV2 capsid was selected for viral production upon completion of a serotype survey in UVM cells. Transduction with rAAV2-shGNAQQ209L induced significant cell death in GNAQQ209L cells but not in a GNAQwt UVM line. Unexpectedly, cell death in the GNAQQ209L UVM cells was also observed upon transduction with the non-targeting control rAAV2 (although to a lesser degree than rAAV2-shGNAQQ209L), suggesting that an element of the AAV vector itself exhibits toxicity in GNAQQ209L UVM cells. This work is among the first describing a genetic-based rAAV approach to specifically target an oncogenic mutant driver allele using single base pair allelic discrimination, collectively demonstrating that both siRNA and rAAV methods of GNAQQ209L depletion result in significant UVM cell death.http://www.sciencedirect.com/science/article/pii/S295032992500089XMT: Regular Issueuveal melanomaadeno-associated virusAAVcancer gene therapyGNAQ |
| spellingShingle | Trace F. McCall Emma J. Sawyer Joshua Darnell Matthew L. Hirsch Jacquelyn J. Bower Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells Molecular Therapy: Oncology MT: Regular Issue uveal melanoma adeno-associated virus AAV cancer gene therapy GNAQ |
| title | Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells |
| title_full | Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells |
| title_fullStr | Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells |
| title_full_unstemmed | Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells |
| title_short | Allele-specific depletion of GNAQQ209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQQ209L uveal melanoma cells |
| title_sort | allele specific depletion of gnaqq209l via sirna or an raav2 shrna vector induces selective toxicity in gnaqq209l uveal melanoma cells |
| topic | MT: Regular Issue uveal melanoma adeno-associated virus AAV cancer gene therapy GNAQ |
| url | http://www.sciencedirect.com/science/article/pii/S295032992500089X |
| work_keys_str_mv | AT tracefmccall allelespecificdepletionofgnaqq209lviasirnaoranraav2shrnavectorinducesselectivetoxicityingnaqq209luvealmelanomacells AT emmajsawyer allelespecificdepletionofgnaqq209lviasirnaoranraav2shrnavectorinducesselectivetoxicityingnaqq209luvealmelanomacells AT joshuadarnell allelespecificdepletionofgnaqq209lviasirnaoranraav2shrnavectorinducesselectivetoxicityingnaqq209luvealmelanomacells AT matthewlhirsch allelespecificdepletionofgnaqq209lviasirnaoranraav2shrnavectorinducesselectivetoxicityingnaqq209luvealmelanomacells AT jacquelynjbower allelespecificdepletionofgnaqq209lviasirnaoranraav2shrnavectorinducesselectivetoxicityingnaqq209luvealmelanomacells |