Compositions and methods for expression of IL-12 and IL-1RA
Inventors
Wang, Nathaniel Stephen • Miyake-Stoner, Shigeki Joseph • Aliahmad, Parinaz
Assignees
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Publication Number
US-11873507-B2
Publication Date
2024-01-16
Expiration Date
Abstract
The present disclosure relates to the field of molecular virology, and particularly relates to nucleic acid molecules encoding a modified alphavirus virus viral genome or self-replicating RNA (srRNA) construct, pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for eliciting a pharmacodynamics effect in a subject.
Core Innovation
The invention relates to a nucleic acid construct comprising a modified alphavirus genome or self-replicating RNA (srRNA) obtained from an alphavirus genome or srRNA, wherein viral structural-protein coding regions are replaced. The replacement includes a polypeptide construct that encodes IL-12A (p35), IL-12B (p40), and an interleukin-1 receptor antagonist (IL-1RA), or functional variants thereof.
The coding sequences for IL-12A, IL-12B, and IL-1RA are operably linked to one another and express protein that is functional in a bioactivity assay. The nucleic acid sequence is further defined by sequence identity to SEQ ID NO: 10, including at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
The disclosure also covers design and expression arrangements using connector strategies, including autoproteolytic cleavage sequences and/or IRES elements, with mono-genic versus multi-genic or polycistronic ORFs and named connector options such as 2A/Furin and IRES elements such as EMCV/KSHV. In addition to the nucleic acid constructs themselves, the disclosure includes formulation and delivery approaches for administering the srRNA vectors, including recombinant cells and pharmaceutical compositions using lipid nanoparticle (LNP), liposomes, viral replicon particles (VRPs), polymer nanoparticles, and other described components.
Claims Coverage
The independent claim requires a modified alphavirus genome or srRNA engineered by replacing viral structural-protein coding sequences with a linked IL-12A (p35), IL-12B (p40), and IL-1RA polypeptide construct, with expression of functional protein in a bioactivity assay and high sequence identity to SEQ ID NO: 10. The inventive features are directed to the engineered alphavirus/srRNA payload and the operable linkage and identity constraints of the IL-12A/IL-12B/IL-1RA coding sequences.
Engineered modified alphavirus genome or srRNA by structural-protein replacement
A nucleic acid construct comprising a modified alphavirus genome or self-replicating RNA (srRNA) obtained from an alphavirus genome or srRNA, wherein viral structural-protein coding sequences are replaced with a coding sequence for a polypeptide construct.
Polypeptide construct encoding IL-12A (p35), IL-12B (p40), and IL-1RA
The polypeptide construct comprises a coding sequence for a p35 subunit of interleukin 12 (p35 or IL-12A) or a functional variant thereof; a coding sequence for a p40 subunit of interleukin 12 (p40 or IL-12B) or a functional variant thereof; and a coding sequence for an interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof.
Operable linkage of IL-12A, IL-12B, and IL-1RA and functional bioactivity expression
The coding sequences for IL-12A, IL-12B, and IL-1RA, or functional variants thereof, are operably linked to one another, and express protein that is functional in a bioactivity assay.
High sequence identity to SEQ ID NO: 10
The nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 10.
Across the independent claim, the coverage centers on an engineered modified alphavirus genome or srRNA in which viral structural-protein coding sequences are replaced by an operably linked IL-12A (p35), IL-12B (p40), and IL-1RA polypeptide construct that expresses functional protein in a bioactivity assay, with the nucleic acid sequence meeting specified sequence-identity thresholds to SEQ ID NO: 10.
Stated Advantages
Expresses protein that is functional in a bioactivity assay.
Documented Applications
Inducing pharmacodynamic effects, including immune response markers like IFN-γ, in subjects with cancer, immune disease, or chronic infection.
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