Adenovirus library and methods
Inventors
Yamamoto, Masato • Miura, Yoshiaki
Assignees
University of Minnesota System
Publication Number
US-12351797-B2
Publication Date
2025-07-08
Expiration Date
2032-04-16
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Abstract
Described herein is a method that generally includes infecting a host cell with a rescue adenovirus, wherein the rescue adenovirus genome comprises a loxP site and encodes at least one marker, and wherein the host cell comprises a library of polynucleotides that complement the adenovirus genome marker and encode a detectable polypeptide; incubating the infected host cell under conditions effective to permit recombination between the adenovirus genome and one or more of the library polynucleotides and the production of recombinant adenovirus particles comprising at least on detectable polypeptide; and detecting the at least one detectable polypeptide. Also described are adenovirus libraries constructed using such a method.
Core Innovation
The invention describes a method for producing recombinant adenovirus libraries with high diversity by infecting host cells with a rescue adenovirus genome that contains a loxP site and at least one marker, while the host cells contain a library of polynucleotides that complement the adenovirus genome marker and encode detectable polypeptides. Recombination occurs between the adenovirus genome and these library polynucleotides, leading to the production of recombinant adenovirus particles possessing detectable polypeptides, which can then be identified.
The problem solved by the invention arises from the very low conversion efficiency from virus-coding plasmids to infectious adenovirus particles in conventional methods, which limits the ability to generate adenovirus libraries with sufficient diversity for applications such as cDNA library work and fiber library screening for targeting motifs. The invention addresses low plasmid-to-virus conversion inefficiency, which historically restricts library sizes to around 10^6 diversity, insufficient for certain therapeutic and targeting applications.
The method involves the use of a fiberless, genetically engineered rescue adenovirus with a single loxP site, and a library of shuttle plasmids each containing a loxP site and library sequences, with both introduced into CRE-expressing vector producer cells. This system allows efficient Cre-lox recombination in producer cells, yielding fiber-modified adenovirus libraries with an unprecedented diversity on the order of 10^10, significantly surpassing previous methods. The invention further exemplifies the method’s application to generate infectious, targeted adenoviruses with selected fiber modifications for selective binding and replication in specific tumor cells, notably mesothelin-expressing cancer cells.
Claims Coverage
The patent includes two independent claims that cover an adenovirus with a specific AB-loop sequence and methods of delivering anti-tumor therapy using this adenovirus. The inventive features focus on the virus composition, targeting ability, and therapeutic application.
Adenovirus with AB-loop comprising VTINRSA (SEQ ID NO: 12)
The claim covers an adenovirus incorporating an AB-loop that includes the amino acid sequence VTINRSA, which confers selective targeting properties, particularly binding to mesothelin-expressing tumor cells.
Method of delivering anti-tumor therapy using the adenovirus with AB-loop VTINRSA
A method comprising administering to a subject the adenovirus with the VTINRSA AB-loop, facilitating binding to mesothelin-expressing tumor cells and subsequent infection, thereby delivering anti-tumor therapy.
Anti-tumor therapy involving virus replication and tumor cell lysis
The anti-tumor therapy includes replication of the administered adenovirus within the target tumor cell, leading to lysis of the tumor cell.
Anti-tumor therapy delivering a therapeutic polynucleotide
The method involves delivery of a therapeutic polynucleotide via the adenovirus to the tumor cell, and expression of this polynucleotide by the tumor cell.
Use against specific tumor cell types
The claims specify the adenovirus and methods are applicable to pancreatic tumor cells and lung carcinoma cells.
Systemic administration of the adenovirus
The adenovirus can be administered systemically to the subject to reach and infect tumor cells expressing mesothelin.
The claims encompass a genetically modified adenovirus featuring a novel AB-loop sequence that enables selective targeting to mesothelin-expressing tumor cells, as well as methods using this adenovirus for delivering anti-tumor therapy through infection, replication, and delivery of therapeutic agents, applicable to pancreatic and lung cancers, including systemic administration.
Stated Advantages
The method enables generation of adenovirus libraries with diversity on the order of 10^10, surpassing conventional methods by several orders of magnitude.
The system significantly improves plasmid-to-virus conversion efficiency, allowing rapid vector production within approximately 48 hours.
The approach allows identification of targeting motifs that confer selective binding and replication in target tumor cells.
The method facilitates generation of adenovirus vectors less prone to mutation compared to conventional plaque-based vector generation.
Resultant fiber-modified adenoviruses exhibit reduced off-target organ distribution (notably liver) and increased tumor tissue distribution in vivo after systemic administration.
Documented Applications
Identification of motifs in target cells that confer specific adenovirus attachment and infection.
Identification of adenovirus vector structures suitable for systemic targeting to tumors, including modifications reducing liver sequestration.
Generation of adenovirus cDNA libraries enabling high throughput drug screening and identification of disease-relevant genes.
Rapid production of adenovirus vectors with enhanced diversity and stability for targeted gene delivery and oncolytic virus development.
Development of infectivity-selective oncolytic adenoviruses (ISOAd) for selective tumor cell entry and replication, particularly targeting mesothelin-expressing cancers such as pancreatic and lung cancers.
Use of adenovirus vectors carrying therapeutic polynucleotides for anti-tumor gene therapy applications.
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