Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models.

Area of Science:

• Gene therapy
• Viral vectors
• Oncology

Background:

• Toca 511, a retroviral replicating vector (RRV), utilizes an internal ribosomal entry site (IRES) for expressing yeast cytosine deaminase (yCD2) to convert 5-fluorocytosine into 5-fluorouracil.
• The IRES element, approximately 600 bp, imposes limitations on the RRV genome size, restricting the incorporation of additional therapeutic transgenes.
• Genetic stability of the RRV-IRES-yCD2 system has been confirmed in preclinical and clinical settings.

Purpose of the Study:

• To overcome RRV genome size limitations by replacing the IRES element with 2A peptides.
• To evaluate the efficiency of 2A peptides, with and without a GSG linker, in expressing multiple transgenes from the RRV platform.
• To compare the therapeutic efficacy of the RRV-IRES-yCD2 and RRV-g2A-yCD2 systems in a preclinical intracranial tumor model.

Main Methods:

• The IRES sequence in the RRV vector was replaced with 2A peptides derived from picornaviruses, with variations including a GSG linker.
• The efficiency of polyprotein separation and transgene expression was assessed for different 2A peptide configurations.
• The RRV vector's capacity to accommodate single or dual transgene insertions using 2A peptides was investigated.
• Comparative efficacy studies were conducted using RRV-IRES-yCD2 and RRV-g2A-yCD2 vectors in an intracranial tumor mouse model.

Main Results:

• The GSG-linked 2A (g2A) peptide demonstrated superior polyprotein separation efficiency compared to non-linked 2A peptides.
• The RRV vector successfully tolerated the insertion of two separate 2A peptides, enabling dual transgene expression without compromising viral assembly or function.
• The RRV-g2A-yCD2 configuration resulted in higher yCD2 protein activity compared to the RRV-IRES-yCD2 system.
• Mice treated with the RRV-g2A-yCD2 vector exhibited a significant survival benefit in the intracranial tumor model.

Conclusions:

• Replacing IRES with 2A peptides, particularly the g2A variant, enhances transgene expression and therapeutic efficacy of RRV vectors.
• The RRV platform's capacity for dual transgene expression using 2A peptides expands the potential for developing novel oncolytic পণ্য candidates.
• These findings support the advancement of RRV-based gene therapies with improved therapeutic outcomes and broader applicability.