Picornavirus RNA Genome Production with Ribozyme-Cleaved Native Ends
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Solution Overview
Problem
Clinical use of replication-competent viruses for cancer treatment is limited by innate and adaptive immune responses, including neutralizing antibodies, which prevent viral lysis of target cells and render re-administration ineffective, especially in metastatic cancers.
Innovation Solution
Development of recombinant DNA molecules with 5′ and 3′ junctional cleavage sequences, including ENV27 ribozymes, to produce synthetic RNA viral genomes with native ends, encapsulated in lipid nanoparticles for efficient delivery and replication in target cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If replication-competent viruses are used for cancer treatment, then direct tumor cell lysis and immune response generation are achieved, but neutralizing antibodies prevent re-administration and limit efficacy in metastatic cancers
Solution Approach 1:
The viral genome is segmented into two separate components: (1) a replication-competent viral genome that provides lytic activity, and (2) a separate polynucleotide encoding a viral antigen. This segmentation allows the antigen to be delivered without the entire virus, enabling re-administration in patients who have developed neutralizing antibodies against the native virus.
Solution Approach 2:
The viral antigen encoding sequence is extracted from the complete viral genome and delivered separately via lipid nanoparticle. This extraction allows the antigen to be administered independently, bypassing the neutralizing antibody barrier that would otherwise prevent re-administration of the whole virus.
2Adaptability or versatility
If artificial particles are used to deliver polynucleotides, then neutralizing antibody issues are avoided, but efficient production of recombinant polynucleotides with native viral ends remains challenging
Solution Approach 1:
A bacteriophage T7 RNA polymerase promoter is introduced as an intermediary element upstream of the viral antigen encoding sequence. This promoter enables efficient in vitro transcription of the recombinant polynucleotide, simplifying the manufacturing process while maintaining the ability to produce polynucleotides with native viral ends.
Solution Approach 2:
The polynucleotide is designed with specific parameters including a 5' cap structure, 5' UTR containing an internal ribosome entry site (IRES), and 3' UTR with polyadenylation signals. These parameter changes optimize the polynucleotide for both efficient in vitro production and effective translation in host cells.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables safe and efficacious systemic delivery of recombinant RNA molecules that can lyse cancer cells and induce adaptive immune responses, effectively treating a range of proliferative disorders including various cancers.
Implementation Method 1
the 5′ junctional cleavage sequence comprises or consists of a ENV27 ribozyme encoding sequence
Implementation Method 2
encapsulated in lipid nanoparticles for efficient delivery and replication in target cells
Data Source
AI summary
The present disclosure relates to production of recombinant RNA molecules encoding an oncolytic virus genome, such as a picornavirus, with native 5′ end of the viral genome utilizing ribozymes. The present disclosure further relates to the design of corresponding DNA template and the use of the recombinant RNA molecules and/or corresponding particles for the treatment and prevention of cancer.