Methods and compositions using auxotrophic regulatable cells
AssigneesAuxolytic Ltd, The Board of Trustees of the Leland Stanford Junior University
InventorsJames Patterson, Matthew Porteus, Volker Wiebking
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Abstract
The present disclosure provides compositions and methods for producing and using modified auxotrophic host cells for improved therapy involving administration of an auxotrophic factor.
Claims124
A donor template comprising:
- (a) one or more nucleotide sequences homologous to a region of an auxotrophy-inducing locus, or homologous to the complement of said region of the auxotrophy-inducing locus, and
- (b) a transgene encoding a therapeutic factor, optionally linked to an expression control sequence.
A vector comprising:
- (a) one or more nucleotide sequences homologous to a region of the auxotrophy-inducing locus, or homologous to the complement of said region of the auxotrophy-inducing locus, and
- (b) a transgene encoding a therapeutic factor.
The donor template or vector of any one of the preceding claims, wherein the transgene is flanked on both sides by the nucleotide sequences homologous to a region of the auxotrophy-inducing locus or the complement thereof.
The donor template or vector of any one of the preceding claims, wherein the auxotrophy-inducing locus is a gene encoding a protein that is involved in synthesis, recycling or salvage of an auxotrophic factor.
The donor template or vector of any one of the preceding claims, wherein the auxotrophy-inducing locus is within a gene in Table 1 or within a region that controls expression of a gene in Table 1.
The donor template or vector of any one of the preceding claims, wherein the auxotrophy-inducing locus is within a gene encoding uridine monophosphate synthetase (UMPS).
The donor template or vector of any one of the preceding claims, wherein the auxotrophy-inducing locus is within a gene encoding holocarboxylase synthetase.
The donor template or vector of any one of the preceding claims, wherein the nucleotide sequence homologous to a region of the auxotrophy-inducing locus is 98% identical to at least 200 consecutive nucleotides of the auxotrophy-inducing locus.
The donor template or vector of any one of the preceding claims, wherein the nucleotide sequence homologous to a region of the auxotrophy-inducing locus is 98% identical to at least 200 consecutive nucleotides of human uridine monophosphate synthetase or holocarboxylase synthetase or any of the genes in Table 1.
The donor template or vector of any one of the preceding claims, further comprising an expression control sequence operably linked to said transgene.
The donor template or vector of any one of the preceding claims, further comprising a marker gene.
The donor template or vector of any one of the preceding claims, wherein the transgene encodes a protein selected from the group consisting of hormones, cytokines, chemokines, interferons, interleukins, interleukin-binding proteins, enzymes, antibodies, Fc fusion proteins, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, receptors, cell surface antigens, receptor antagonists, and co-stimulating factors, structural proteins, cell surface antigens, ion channels, an epigenetic modifier, and an RNA editing protein.
The donor template or vector of any one of the preceding claims, wherein the transgene encodes a T cell antigen receptor.
A nuclease system for targeting integration of a transgene to an auxotrophy-inducing locus comprising:
- (a) a Cas9 protein, and
- (b) a guide RNA specific for an auxotrophy-inducing locus.
A nuclease system for targeting integration of a transgene to an auxotrophy-inducing locus comprising:
- a meganuclease specific for said auxotrophy-inducing locus.
A modified host cell ex vivo, comprising:
- a transgene encoding a therapeutic factor integrated at an auxotrophy-inducing locus, wherein said modified host cell is auxotrophic for an auxotrophic factor and capable of expressing the therapeutic factor.
A kit containing said donor template or vector or nuclease system or modified host cell, or a combination thereof, of any one of the preceding claims, optionally with a container or vial.
A method of alleviating or treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject:
- (a) a composition comprising a modified host cell comprising a transgene encoding a protein integrated at an auxotrophy-inducing locus, wherein the modified host cell is auxotrophic for an auxotrophic factor;
- and
- (b) the auxotrophic factor in an amount sufficient to produce therapeutic expression of the protein.
A modified host cell ex vivo, comprising:
- a transgene encoding a therapeutic factor, wherein said modified host cell is auxotrophic for an auxotrophic factor and capable of expressing the therapeutic factor.
A modified T cell, comprising:
- a knockout of the UMPS gene, rendering the modified T cell auxotrophic for a uracil source or uridine;
- and a transgene encoding a CAR.
A method of producing a modified mammalian host cell comprising:
- (a) introducing into said mammalian host cell one or more nuclease systems that targets and cleaves DNA at an auxotrophy-inducing locus, or a nucleic acid encoding one or more components of said one or more nuclease systems;
- and (b) introducing into said mammalian host cell a donor template encoding a therapeutic factor.
Dependencies are inferred from the claim text.