Non-toxic HSV Vectors for Persistent Gene Delivery
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Solution Overview
Problem
There is a need for a herpes simplex virus (HSV) vector that can express transgenes in various tissues or cells without causing harm, and that can persistently express transgenes for an extended period without integrating into the host genome.
Innovation Solution
The development of an HSV vector that does not express toxic viral genes in non-complementing cells, capable of persistent transgene expression for at least 28 days, and utilizes insulator sequences to protect transgene expression from silencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If HSV vectors retain essential immediate-early genes (ICP0, ICP4, ICP27) for viral replication, then the virus can replicate and produce progeny virions, but the vectors become toxic to cells and cause cytotoxicity
Solution Approach 1:
The patent extracts and removes the harmful immediate-early genes (ICP0, ICP4, ICP27, ICP22, ICP47) from the HSV vector genome, creating a replication-defective vector that cannot produce toxic viral proteins in non-complementing cells, thereby eliminating cytotoxicity while maintaining transgene expression capability
Solution Approach 2:
The patent introduces complementing cells as intermediary producers that express the essential viral genes (ICP4, ICP27) needed for vector replication. These complementing cells serve as a bridge, allowing the vector to replicate without expressing toxic genes in the target tissue, thus separating the replication function from the harmful effects
2Object-affected harmful factors
If HSV vectors delete essential immediate-early genes (ICP0, ICP4, ICP27) to eliminate cytotoxicity, then the virus becomes non-toxic in non-complementing cells, but the vectors lose the ability to replicate and produce progeny virions
Solution Approach 1:
The patent employs complementing cells as intermediary producers that supply the essential viral proteins (ICP4, ICP27) needed for vector replication. This allows the vector to replicate without retaining the toxic immediate-early genes in its genome, thus maintaining both non-toxicity and replication capability through a separation of functions between vector and host cell
Solution Approach 2:
The patent segments the viral replication function from the vector genome by using complementing cells to provide the essential replication proteins. This segmentation allows the vector to be non-toxic (lacking ICP0, ICP4, ICP27) while still being able to replicate through the complementary proteins supplied by the host cell
3Object-affected harmful factors
If HSV vectors are designed to be replication-defective to ensure safety and non-toxicity, then the vectors cannot produce harmful viral proteins, but the transgene expression may be limited or reduced
Solution Approach 1:
The patent removes the toxic immediate-early genes from the vector while preserving the latency-associated transcript (LAT) region and other essential elements for transgene expression. This extraction of harmful elements maintains vector safety while preserving the ability to drive sustained transgene expression through alternative mechanisms such as LAT promoter activity and insulator sequences
4Productivity
If HSV vectors use strong promoters for transgene expression, then transgene expression levels increase, but the risk of expressing harmful viral genes or causing cytotoxicity increases
Solution Approach 1:
The patent extracts and removes the harmful immediate-early gene promoters and regulatory elements from the vector genome. This removal eliminates the risk of harmful viral gene expression even when strong transgene promoters are used, as the toxic promoters have been physically deleted from the vector architecture
Solution Approach 2:
The patent segments the regulatory functions by separating the transgene promoter from any potential viral promoter activity. The transgene is driven by its own promoter while the vector lacks the harmful immediate-early promoters, creating a clear functional separation that prevents cross-activation of toxic genes
Data Source
AI summary
Disclosed is a method for administering a transgene into a fibroblast in a subject comprising: a) providing a herpes simplex virus (HSV) comprising a recombinant herpes simplex virus genome, wherein said recombinant herpes simplex virus genome comprises one or more transgenes encoding a polypeptide to be expressed in said fibroblast; and b) providing a pharmaceutically acceptable carrier, wherein said HSV has reduced cytotoxicity as compared to a wild-type herpes simplex virus.