Alpharetrovirus Particles for Nonintegrating Transient RNA Delivery

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Solution Overview

Problem

Existing retroviral vectors face challenges in efficiently transducing animal cells, particularly human cells, while avoiding insertional mutagenesis and cytotoxicity from stable transgene expression, especially with gene editing tools like SpCas9, Cre recombinase, and Sleeping Beauty DNA transposase.

Innovation Solution

Alpharetrovirus-based particles are developed, comprising a protein component with specific domains and an MS2 bacteriophage-derived coat protein dimer, which efficiently package and deliver RNA constructs, including coding and non-coding RNAs, without integrative mutagenesis, using a pseudotyping protein for target specificity and protease sites for domain separation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If retroviral vectors are used to deliver RNA constructs into animal cells, then transduction efficiency is improved, but insertional mutagenesis and cytotoxicity occur due to stable transgene expression

Engineering Contradiction:

Improvetransduction efficiencyVSAvoidinsertional mutagenesis and cytotoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent employs alpharetrovirus-based particles that deliver RNA constructs for transient expression rather than stable integration. The RNA molecules are non-integrating and naturally degraded over time, providing temporary transduction without permanent genetic modification. This disposable approach allows high transduction efficiency while avoiding the harmful effects of stable integration, as the RNA constructs are replaced and degraded naturally without causing insertional mutagenesis or prolonged cytotoxicity

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The patent extracts the integration function from the retroviral vector system by using alpharetrovirus-based particles that deliver naked RNA constructs instead of integrating DNA. The RNA molecules are packaged in particles but do not integrate into the host genome, separating the transduction function from the integration function. This extraction eliminates insertional mutagenesis while maintaining efficient cellular delivery of the therapeutic RNA constructs

Inventive Principle:

Principle #2Taking out (Extraction)

2Adaptability or versatility

If multiple RNA constructs are delivered simultaneously, then spatiotemporal co-delivery capability is improved, but particle complexity increases

Engineering Contradiction:

Improvespatiotemporal co-delivery capabilityVSAvoidparticle complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The alpharetrovirus-based particle system serves multiple functions simultaneously: it packages multiple different RNA constructs (coding and non-coding RNAs), delivers them to target cells, and enables transient expression of multiple genes. The particle structure is universal and can accommodate various RNA types including CRISPR/Cas9 components, shRNA, and mRNA constructs, providing multi-functionality without requiring separate delivery systems for each RNA type

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent merges multiple RNA constructs into a single alpharetrovirus-based particle system. Different RNA molecules (e.g., Cas9 mRNA, sgRNA, and other therapeutic RNAs) are co-packaged within the same viral particles, enabling simultaneous delivery to target cells. This combining approach simplifies the delivery system compared to using separate vectors for each RNA construct, while maintaining the ability to achieve complex spatiotemporal co-delivery patterns

Inventive Principle:

Principle #5Merging (Combining)

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

The alpharetrovirus-based particles achieve high-efficiency, transient transduction of animal cells, protecting RNA integrity and reducing cytotoxicity, enabling spatiotemporal co-delivery of RNA constructs for gene editing and expression.

Implementation Method 1

comprising a protein component with specific domains and an MS2 bacteriophage-derived coat protein dimer, which efficiently package and deliver RNA constructs

Methodology Applied

Scientific EffectProtein-RNA binding:

Implementation Method 2

using a pseudotyping protein for target specificity

Methodology Applied

Scientific EffectProtein-ligand binding:

Implementation Method 3

and protease sites for domain separation

Methodology Applied

Scientific EffectProteolytic cleavage: Enzyme

Data Source

PatentUS20250333759A1Alpharetrovirus-based particles for delivery of RNA into cells

Publication Date: 2025.10.30 MEDIZINISCHE HOCHSCHULE HANNOVER

AI summary

The new alpharetrovirus-based particles are suitable for high efficiency of transiently transducing animal cells. e.g. human or murine cells, and which efficiently introduce coding and non-coding RNA contained in the alpharetrovirus-based particles into target cells The alpharetrovirus-based particles also provide for high efficiency of the activity and/or integrity of the RNA that is introduced into the animal cells, as the particles protect the incoming RNA from degradation during entry. The transferred RNA can be of non-coding nature (e.g. single guide (sg) RNA. short-hairpin (sh) RNA. micro RNA. or long non-coding (Inc) RNA. or the RNA may encode proteins or peptides. e.g. receptors. transcription factors. cellular enzymes, antigens for use in vaccination, gene/protein therapy and/or gene editing nucleases, recombinases and transposases.