Ungulates with genetically modified immune systems
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Publication Number
US-9585374-B2
Publication Date
2017-03-07
Expiration Date
Abstract
The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunogobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.
Core Innovation
The invention relates to transgenic porcine animals and targeting constructs for immunoglobulin gene loci, including disruption of porcine immunoglobulin regions using 5′ and 3′ recombination arms homologous to specific constant or joining-region sequences. The approach includes selectable markers and homologous recombination-based modification of endogenous immunoglobulin regions at the DNA level, with substitution, deletion, or insertion techniques.
For the porcine heavy chain joining region (J region), the invention specifies a homozygous disruption of nucleotides 3296-3352 of SEQ ID NO: 29, such that the porcine lacks expression of the endogenous porcine heavy chain immunoglobulin gene. The disclosure also describes additional disruption strategies for kappa and lambda regions, including lambda constant-region targeting and a two-step lambda J/C deletion strategy using lox sites and Cre recombinase.
The disclosed subject matter further provides novel genomic sequences for the porcine lambda light chain locus of ungulate immunoglobulins, including locus organization, isolated porcine lambda nucleotide sequences, flanking sequence, and an enhancer region. It also describes engineered ungulates in which human immunoglobulin loci or xenogenous immunoglobulin loci are introduced on artificial chromosomes or by endogenous integration, with expression of diverse human antibodies after antigen exposure.
Claims Coverage
The independent claim coverage centers on a transgenic porcine animal with a homozygous disruption of the endogenous porcine heavy chain joining region (J region) that eliminates endogenous heavy chain immunoglobulin expression. Across the provided claims, there are 6 inventive features, with dependent refinements covering the disruption method, lambda constant-region disruption, transcription without translation outcomes, and xenogenous immunoglobulin embodiments.
Homozygous disruption of the endogenous porcine heavy chain J region
A transgenic porcine animal whose genome comprises a homozygous disruption of nucleotides 3296-3352 of SEQ ID NO: 29 of the joining region (J region) of the endogenous porcine heavy chain gene, wherein the porcine lacks expression of the endogenous porcine heavy chain immunoglobulin gene.
Homozygous heavy chain J region disruption produced by homologous recombination
The homozygous disruption described in the transgenic porcine animal is produced by homologous recombination.
Heavy chain gene disruption by substitution, deletion, or insertion
The porcine heavy chain gene is disrupted by substitution, deletion, or insertion techniques.
Lambda constant region disruption eliminates endogenous lambda expression
A transgenic porcine animal having a homozygous disruption of the constant region of the endogenous porcine lambda immunoglobulin gene such that this disruption eliminates expression of the endogenous gene.
Transcription without translation for immunoglobulin genes
A porcine construct characterized such that its porcine heavy chain, kappa light chain, and lambda light chain genes can be transcribed into RNA but cannot be translated into protein.
Xenogenous human immunoglobulin in the transgenic subject
The xenogenous immunoglobulin is a human immunoglobulin or a fragment of it.
The claim coverage focuses on genetically engineered porcine animals with homozygous disruption of defined endogenous immunoglobulin regions, especially the heavy-chain J region, to prevent endogenous heavy-chain immunoglobulin expression. Dependent coverage further includes lambda locus disruption, specified disruption techniques, transcription without translation outcomes, and xenogenous immunoglobulin embodiments, including human immunoglobulin or fragments of it.
Stated Advantages
The porcine lacks expression of the endogenous porcine heavy chain immunoglobulin gene.
The disruption of the constant region of the endogenous porcine lambda immunoglobulin gene eliminates expression of the endogenous lambda gene.
The porcine construct enables transcription into RNA while preventing translation into protein for heavy chain, kappa light chain, and lambda light chain genes.
Documented Applications
Engineered ungulates, including pigs, in which disruption of porcine immunoglobulin loci and introduction of human or xenogenous immunoglobulin loci on artificial chromosomes is described in the context of expressing diverse human antibodies after immunization or antigen exposure.
Generating genetically modified ungulates using nuclear transfer and related production approaches.
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