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Publication Number
US-12312593-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Provided herein are lentiviral vectors comprising a mutated, heterologous envelope protein, a targeting protein, and at least one transgene for delivery to and expression by a cell characterized by the targeting protein. Also provided are methods and materials for producing the lentiviral vectors described herein, methods for transducing target cells, and cells transduced by lentiviral vectors according to the present disclosure.
Core Innovation
The invention describes engineered self-inactivating lymphocyte-targeted lentiviral vectors (LVVs) that deliver a transgene, with an option for a chimeric antigen receptor (CAR). The vectors use a viral envelope that combines a mutated heterologous fusogenic envelope protein with lymphocyte targeting functionality, configured to provide lymphocyte targeting while reducing LDL receptor binding and retaining fusogenic fusion properties.
A mutated heterologous fusogenic envelope protein is provided, exemplified by VSV-G variants and related sequence variants in which specific amino acid substitutions reduce LDL receptor binding while retaining fusion. The same envelope also displays a membrane-bound lymphocyte targeting protein that includes an extracellular lymphocyte-targeting domain and a transmembrane domain, including CD80 targeting and anti-CD3 constructs.
The LVVs further include embodiments in which the targeting protein and the mutated VSV-G are expressed from a single env plasmid using tandem expression cassettes, as well as multi-plasmid and five-plasmid packaging formats with VSV-G env plasmid, GagPol plasmid, Rev plasmid, and an additional targeting protein plasmid. The disclosure also describes lentiviral packaging systems in a third-generation format with self-inactivating design using U3 deletion in the 3' LTR, and CAR embodiments in which the transgene includes ITAM-containing intracellular signaling domains and costimulatory domains such as 4-1BB, with examples including anti-CD19 and anti-BCMA CARs.
Claims Coverage
The claim coverage includes 5 inventive features.
Polypeptide of SEQ ID NO: 90 with substitution at position 182
A polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 90, wherein the polypeptide comprises an amino acid substitution at position 182.
Constrained substitutions for position 352
The polypeptide comprises an amino acid substitution at position 352 where threonine is replaced by one amino acid selected from a specified set of residues.
Nucleic acid encoding the polypeptide
A nucleic acid encoding the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 90 with the substitution at position 182.
Lentiviral vector envelope with heterologous lymphocyte targeting protein
A lentiviral vector whose envelope includes a heterologous lymphocyte targeting protein comprising an extracellular lymphocyte targeting domain and a transmembrane domain.
Lentiviral vector with expression cassette encoding a heterologous CAR transgene
A lentiviral vector includes an expression cassette encoding a heterologous chimeric antigen receptor (CAR) transgene.
The claim coverage is centered on a specifically defined polypeptide (SEQ ID NO: 90 with substitutions at position 182 and further constrained substitutions at position 352), and extends to an encoding nucleic acid and to lentiviral vector embodiments. The lentiviral vector embodiments include an envelope with a heterologous lymphocyte targeting protein and an expression cassette encoding a heterologous CAR transgene.
Stated Advantages
Reduced LDL receptor binding while retaining fusogenic fusion properties.
Provides lymphocyte targeting.
Self-inactivating design using U3 deletion in the 3' LTR.
Tropism-redirected LVV can achieve high-titer production.
Preferential transduction of T cells over B cells.
Reduced need for exogenous T cell activation.
Improved CAR-T generation in PBMC mixtures.
Documented Applications
Delivery of a transgene.
Delivery of a chimeric antigen receptor (CAR) transgene.
Anti-CD19 and anti-BCMA CARs.
CD80 targeting and anti-CD3 constructs.
Use in engineered lymphocytes, including CAR-modified T cells, where tropism-redirected LVV preferentially transduces T cells over B cells.
Application to CAR-T generation from PBMC mixtures, with reduced need for exogenous T cell activation.
Tropism assays comparing transduction in T-cell versus B-cell contexts are referenced to support preferential targeting.
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