Epidermal growth factor receptor (EGFR) and methods of use in adenoviral-associated virus type 6 (AAV6) transduction
Inventors
Chiorini, John • Schmidt, Michael • Weller, Melodie L.
Assignees
US Department of Health and Human Services
Publication Number
US-8808684-B2
Publication Date
2014-08-19
Expiration Date
2030-09-10
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Abstract
Comparative gene analysis (CGA) was combined with pathway visualization software to identify a positive correlation between AAV6 transduction and epidermal growth factor receptor (EGFR) expression. It was found that EGFR is necessary for vector internalization and functions as a co-receptor for AAV6. The identification and characterization of AAV6's requirement of EGFR expression for high transduction activity has allowed construction of recombinant AAV6 vectors which are capable of targeting and killing specific types of head and neck tumors that because of this high EGFR activity, were until now, refractory to current therapies.
Core Innovation
Infection by Adeno-associated virus type 6 (AAV6) requires the epidermal growth factor receptor (EGFR) as a co-receptor for efficient vector internalization in mammalian cells. The present invention identifies and characterizes EGFR as necessary for AAV6 transduction, establishing that EGFR expression correlates positively with AAV6 transduction efficiency based on comparative gene analysis (CGA) and pathway visualization software.
The invention enables the construction of recombinant AAV6 vectors that leverage the requirement for EGFR expression to target and kill specific tumor types, particularly those in the head and neck region, which express high levels of EGFR and are refractory to current therapies. The recombinant AAV6 vector carries heterologous nucleic acid sequences capable of being expressed by the host cell, where the nucleic acid can encode proteins that increase susceptibility to prodrugs or cytotoxic agents, such as suicide genes enhancing tumor cell death.
Claims Coverage
The patent contains a single independent claim describing a method for specifically transducing EGFR-expressing cells using a recombinant AAV6 vector. This claim covers several inventive features regarding vector composition, targeting specificity, and functional outcomes.
Specific transduction of EGFR-expressing cells using recombinant AAV6 vector
A method for introducing a recombinant adeno-associated virus vector comprising the AAV6 viral genome—or a functional portion thereof—directly to cells within a population, selectively transducing EGFR-expressing cells while excluding non-EGFR-expressing cells.
Vector encodes heterologous nucleic acid increasing susceptibility to drug or cytotoxic agent
The recombinant AAV6 vector contains a heterologous nucleic acid sequence capable of expression in EGFR-expressing cells, encoding a gene that increases the cell's susceptibility to a drug or cytotoxic agent, enabling targeted cell killing.
High vector genome copy number in transduced cells
Transduced EGFR-expressing cells specifically contain approximately 4.6×10^4±0.1×10^4 copies of the vector genome per milligram of tissue, indicating efficient vector uptake and persistence.
Heterologous nucleic acid includes nucleic acid or polypeptide encoding, including suicide genes
The heterologous nucleic acid may encode RNA or DNA molecules, polypeptides, or specific genes such as E. coli nitroreductase, cytosine deaminase, Varicella Zoster-tk, Cytochrome P450 B1, carboxypeptidase G2, E. coli purine nucleoside phosphorylase, and notably Herpes Simplex Virus thymidine kinase (HSV-tk), facilitating prodrug activation and cytotoxicity.
Method applicable to in vivo transduction of mammalian tumor cells
The transduction method is performed in vivo, targeting mammalian EGFR-expressing cells, including tumor cells derived from head or neck cancers.
Use of nucleoside analog drugs such as ganciclovir with HSV-tk gene
When the heterologous nucleic acid encodes HSV-tk, the method includes administration of nucleoside analog drugs, e.g., ganciclovir, which become cytotoxic upon enzymatic modification, thereby killing the targeted cells.
The inventive claims cover the use of recombinant AAV6 vectors that specifically transduce EGFR-expressing cells by exploiting the requirement of EGFR for viral internalization, delivering heterologous nucleic acids that sensitize the target cells to cytotoxic agents, with applications in vivo against mammalian tumors, notably head and neck cancers.
Stated Advantages
The identification of EGFR as a co-receptor necessary for AAV6 internalization enables construction of vectors with high specificity for EGFR-expressing cells.
Recombinant AAV6 vectors can target and kill tumor types with high EGFR activity that are refractory to current therapies.
Use of AAV6 vectors results in effective delivery of therapeutic genes resulting in significant reduction in tumor growth in xenograft models.
Documented Applications
Gene therapy targeting mammalian cells expressing EGFR by delivering recombinant AAV6 vectors encoding therapeutic genes.
Selective transduction and killing of cancer cells, particularly head and neck tumors with high EGFR expression, using gene-directed enzyme prodrug therapy with genes such as HSV-tk.
In vivo delivery of therapeutic nucleic acids to tumors by intratumoral injection, convection-enhanced infusion, or intravascular administration.
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