Targeted CRISPR-Cas VLP Delivery for Lower Off-Target Editing
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Solution Overview
Problem
Current delivery strategies for CRISPR-Cas genome editing components, such as recombinant viruses and ribonucleoproteins, lead to prolonged expression in target cells, increasing the likelihood of off-target gene editing events.
Innovation Solution
Virus-like particles (VLPs) comprising a CRISPR-Cas effector polypeptide and a recombinant lentivirus with pseudotyping viral glycoproteins for targeted delivery, allowing for precise genome editing without prolonged expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant viruses are used to deliver CRISPR-Cas components, then delivery efficiency is improved, but CRISPR-Cas effector polypeptide expression duration increases leading to off-target effects
Solution Approach 1:
The delivery system is segmented into two separate components: VLPs that deliver the CRISPR-Cas effector polypeptide and a separate recombinant lentivirus that delivers only the guide RNA expression cassette. This segmentation allows the CRISPR-Cas effector to be delivered transiently without prolonged expression, while the guide RNA is delivered separately to ensure sustained targeting capability.
Solution Approach 2:
The invention extracts the CRISPR-Cas effector polypeptide delivery from the viral vector system and places it into VLPs instead. This extraction removes the problematic prolonged expression characteristic of viral vectors while maintaining delivery efficiency, as VLPs are non-replicative and do not integrate into the host genome.
2Reliability
If viral vectors are used for delivery, then transgene delivery capability is improved, but off-target gene editing events increase
Solution Approach 1:
The invention uses VLPs as disposable, non-replicative delivery vehicles that transduce the target cells but do not establish persistent infection. The VLPs deliver the CRISPR-Cas effector polypeptide and then are degraded, leaving no persistent viral genome to drive prolonged expression. This transient delivery eliminates the source of prolonged off-target effects while maintaining effective initial delivery.
Solution Approach 2:
The invention introduces VLPs as an intermediary delivery system between the CRISPR-Cas components and the target cells. These VLPs serve as a temporary mediator that delivers the effector polypeptide without establishing permanent presence in the cell, unlike integrating viral vectors. The intermediary VLP system thus enables delivery while preventing the harmful prolonged expression that causes off-target effects.
3Object-generated harmful factors
If ribonucleoprotein complexes are used for delivery, then off-target effects are reduced, but delivery efficiency and targeted delivery capability decrease
Solution Approach 1:
The invention changes the physical and functional parameters of the delivery system by using VLPs with pseudotyping viral glycoproteins. This parameter change enables the delivery complex to recognize and bind to specific target cell surface receptors, providing targeted delivery capability that RNP complexes lack. The VLP structure allows for efficient cellular uptake while maintaining the transient expression profile that reduces off-target effects.
Data Source
AI summary
The present disclosure provides virus-like particles (VLPs) comprising: i) a CRISPR-Cas effector polypeptide; ii) a recombinant lentivirus comprising a nucleotide sequence encoding a therapeutic polypeptide having a length of from about 250 amino acids to about 3,000 amino acids, where the VLP comprises a pseudotyping viral glycoprotein and/or a polypeptide that provides for binding to a target cell. The present disclosure provides systems for producing a VLP. The present disclosure provides methods of delivering a therapeutic protein, using a VLP of the present disclosure.