Thymidine kinase gene
Inventors
Levy, John P. • Reed, Rebecca A. • MCNULTY, Joseph • Johnson, Jr., Robert G.
Assignees
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Publication Number
US-10610603-B2
Publication Date
2020-04-07
Expiration Date
Abstract
Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases are provided, including their use in diagnostic and therapeutic applications. The thymidine kinases may be mutated using conservative mutations, non-conservative mutations, or both. Also provided are gene therapeutic systems, including viral and retroviral particles.
Core Innovation
The disclosed invention concerns mutated human herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene products and polynucleotide sequences encoding them. The mutations are defined at specific amino acid residue positions, including residue 32 or 33 or both, with substitutions at residues 32 and 33 corresponding to positions 32 and 33 of SEQ ID NO: 2. The mutation types described include substitutions to an acidic amino acid or to cysteine, and the mutated form increases cell kill activity relative to a wild-type thymidine kinase.
The disclosure further describes additional engineered sequence features and mutation combinations applied to the HSV1-TK coding sequence. Examples include additional mutation sites such as residue 167, along with specified optimized variants, including A168H dmNES/SEQ ID NO:18, A167F, and A167F+A168H. Functional sequence elements are described as being added or localization elements removed or modified, including incorporation of a nuclear export signal, and correction or optimization aspects such as splice donor/acceptor correction and codon optimization relative to SEQ ID NOs.
The disclosure also connects the mutated HSV1-TK sequences to cancer gene therapy use, including delivery using viral or retroviral vector particles encoding the modified HSV1-TK, followed by prodrug administration. Prodrugs described include ganciclovir and valganciclovir, and also acyclovir, valacyclovir, and penciclovir. The document further describes concepts for enhancing therapeutic effect via a bystander effect mediated by gap junction intracellular communication involving connexins or E-cadherin, and provides targeting context including a PiT-2 receptor concept in relation to Reximmune-C2 and related constructs.
Claims Coverage
The document includes at least one independent claim directed to a polynucleotide encoding a mutated HSV1-TK with specific residue 32/33 mutation constraints to increase cell kill activity. Additional dependent claims refine mutation types and add further sequence and delivery features, including a nuclear export signal and use of a retroviral vector for carrying the modified HSV1-TK.
Mutated HSV1-TK polynucleotide for increased cell kill activity
A polynucleotide sequence encoding a mutated form of thymidine kinase from a human herpes simplex virus type 1 (HSV1-TK) for increasing cell kill activity, wherein the encoded HSV1-TK is mutated at either amino acid residue 32 or 33 or both, wherein amino acid residues 32 and 33 are each independently mutated to an acidic amino acid or to cysteine, and wherein the mutated form of thymidine kinase increases cell kill activity relative to a wild-type thymidine kinase.
Residue 32/33 substitution restricted to acidic amino acids
The polynucleotide is such that amino acid residues 32 and 33 are each independently mutated to an acidic amino acid.
Additional mutation at amino acid residue 167
The encoded HSV1-TK protein is further mutated at amino acid residue 167.
Encoded HSV1-TK containing a nuclear export signal
The polynucleotide additionally contains a nuclear export signal in the encoded HSV1-TK sequence.
Retroviral vector carrying the modified HSV1-TK polynucleotide
A retroviral vector includes a polynucleotide encoding a modified HSV1-TK polypeptide.
Overall, the claim coverage centers on polynucleotides encoding mutated HSV1-TK with defined residue 32 and/or 33 substitutions to increase cell kill activity relative to wild-type HSV1-TK, with further refinements describing additional mutation sites, the addition of a nuclear export signal, and retroviral vector delivery carrying the modified HSV1-TK.
Stated Advantages
Increases cell kill activity relative to a wild-type thymidine kinase.
Documented Applications
Cancer gene therapy use with viral or retroviral vector particles encoding modified HSV1-TK, followed by prodrug administration with ganciclovir, valganciclovir, acyclovir, valacyclovir, or penciclovir.
Therapeutic effect enhancement through a bystander effect mediated by gap junction intracellular communication involving connexins or E-cadherin.
Targeting context involving a PiT-2 receptor (SLC20A2) in relation to Reximmune-C2 and related constructs.
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